Porcine cmp-n-acetylneuraminic acid hydroxylase gene

ABSTRACT

The present invention provides porcine CMP-N-Acetylneuraminic-Acid Hydroxylase (CMP-Neu5Ac hydroxylase) protein, cDNA, and genomic DNA regulatory sequences. Furthermore, the present invention includes porcine animals, tissues, and organs, as well as cells and cell lines derived from such animals, tissues, and organs, which lack expression of functional CMP-Neu5Ac hydroxylase. Such animals, tissues, organs, and cells can be used in research and in medical therapy, including in xenotransplantation, and in industrial livestock farming operations.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional patent application Ser. No. 60/476,396, filed Jun. 6, 2003.

FIELD OF THE INVENTION

The present invention provides porcine CMP-N-Acetylneuraminic-Acid Hydroxylase (CMP-Neu5Ac hydroxylase) protein, cDNA, and genomic DNA regulatory sequences. Furthermore, the present invention includes porcine animals, tissues, and organs, as well as cells and cell lines derived from such animals, tissues, and organs, which lack expression of functional CMP-Neu5Ac hydroxylase. Such animals, tissues, organs, and cells can be used in research and in medical therapy, including in xenotransplantation, and in industrial livestock farming operations. In addition, methods are provided to prepare organs, tissues, and cells lacking the porcine CMP-Neu5Ac hydroxylase gene for use in xenotransplantation.

BACKGROUND OF THE INVENTION

The unavailability of acceptable human donor organs, the low rate of long term success due to host versus graft rejection, and the serious risks of infection and cancer are the main challenges now facing the field of tissue and organ transplantation. Because the demand for acceptable organs exceeds the supply, many people die each year while waiting for organs to become available. To help meet this demand, research has been focused on developing alternatives to allogenic transplantation. Dialysis is available to patients suffering from kidney failure, artificial heart models have been tested, and other mechanical systems have been developed to assist or replace failing organs. Such approaches, however, are quite expensive. The need for frequent and periodic access to dialysis machines greatly limits the freedom and quality of life of patients undergoing such therapy.

Xenograft transplantation represents a potentially attractive alternative to artificial organs for human transplantation. The potential pool of nonhuman organs is virtually limitless. Pigs are considered the most likely source of xenograft organs. The supply of pigs is plentiful, breeding programs are well established, and their organ size and physiology are compatible with humans. Therefore, xenotransplantation with pig organs offers a potential solution to the shortage of organs available for clinical transplantation.

Host rejection of such cross-species tissue remains a major concern in this area. The immunological barriers to xenotransplantation have been, and remain, formidable. The first immunological hurdle is “hyperacute rejection” (HAR). HAR is defined by the ubiquitous presence of high titers of pre-formed natural antibodies binding to the foreign tissue. The binding of these natural antibodies to target epitopes on the donor organ endothelium is believed to be the initiating event in HAR. This binding, within minutes of perfusion of the donor organ with the recipient blood, is followed by complement activation, platelet and fibrin deposition, and ultimately by interstitial edema and hemorrhage in the donor organ, all of which cause failure of the organ in the recipient (Strahan, et al. (1996) Frontiers in Bioscience 1, pp. 34-41).

Some noted xenotransplants of organs from apes or old-world monkeys (e.g., baboons) into humans have been tolerated for months without rejection. However, such attempts have ultimately failed due to a number of immunological factors. Even with heavy immunosuppression to suppress HAR, a low-grade innate immune response, attributable in part to failure of complement regulatory proteins (CRPs) within the graft tissue to control activation of heterologous complement on graft endothelium, ultimately leads to destruction of the transplanted organs (Starzl, Immunol. Rev., 141, 213-44 (1994)). In an effort to develop a pool of acceptable organs for xenotransplantation into humans, researchers have engineered animals that produce human CRPs, an approach which has been demonstrated to delay, but not eliminate, xenograft destruction in primates (McCurry, et al., Nat. Med., 1, 423-27 (1995); Bach et al., Immunol. Today, 17, 379-84 (1996)).

In addition to complement-mediated attack, human rejection of discordant xenografts appears to be mediated by a common antigen: the galactose-α(1,3)-galactose (gal-α-gal) terminal residue of many glycoproteins and glycolipids (Galili et al., Proc. Nat. Acad. Sci., (USA), 84, 1369-73 (1987); Cooper, et al., Immunol. Rev., 141, 31-58 (1994); Galili, et al., Springer Sem. Immunopathol, 15, 155-171 (1993); Sandrin, et al., Transplant Rev., 8, 134 (1994)). This antigen is chemically related to the human A, B, and O blood antigens, and it is present on many parasites and infectious agents, such as bacteria and viruses. Most mammalian tissue also contains this antigen, with the notable exception of old world monkeys, apes and humans. (see, Joziasse, et al., J. Biol. Chem., 264, 14290-97 (1989). Individuals without such carbohydrate epitopes produce abundant naturally occurring antibodies (IgM as well as IgG) specific to the epitopes. Many humans show significant levels of circulating IgG with specificity for gal-α-gal carbohydrate determinants (Galili, et al., J. Exp. Med., 162, 573-82 (1985); Galili, et al., Proc. Nat. Acad. Sci. (USA), 84, 1369-73 (1987)). The α-galactosyltransferase (α-GT) enzyme catalyzes the formation of gal-α-gal moieties. Research has focused on the modulation or elimination of this enzyme to reduce or eliminate the expression of gal-α-gal moieties on the cell surface of xenotissue.

The elimination of the α-galactosyltransferase gene from porcine has long been considered one of the most significant hurdles to accomplishing xenotransplantation from pigs to humans. Two alleles in the pig genome encode the α-GT gene. Single allelic knockouts of the α-GT gene in pigs were reported in 2002 (Dai, et al. Nature Biotechnol., 20:251 (2002); Lai, et al., Science, 295:1089 (2002)).

Recently, double allelic knockouts of the α-GT gene have been accomplished (Phelps, et al., Science, 299: pp. 411-414 (2003)). WO 2004/028243 to Revivicor Inc. describes porcine animal, tissue, organ, cells and cell lines, which lack all expression of functional α1,3 galactosyltransferase (α1,3-GT). Accordingly, the animals, tissues, organs and cells lacking functional expression of α1,3-GT can be used in xenotransplantation and for other medical purposes.

PCT patent application WO 2004/016742 to Immerge Biotherapeutics, Inc. describes α(1,3)-galactosyltransferase null cells, methods of selecting GGTA-1 null cells, α(1,3)-galactosyltransferase null swine produced therefrom (referred to as a viable GGTA-1 null swine), methods for making such swine, and methods of using cells, tissues and organs of such a null swine for xenotransplantation.

One of the earliest known xenoantigens other than gal-α-gal is an epitope that Hanganutiu Deicher antibodies recognize, and which have long been associated with serum disease. The epitope has been identified as N-glycolylneuraminic acid (Neu5Gc), a member of the sialic acid family of carbohydrates. Among carbohydrates, sialic acids are abundant and ubiquitous. Sialic acid is a generic designation used for N-acylneuraminic acids (Neu5Acyl) and their derivatives. N-Acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are two of the most abundant derivatives of sialic acids.

The Neu5Gc epitope is located in the terminal position in the glycan chains of glycoconjugates. Due to this exposed position, it plays an important role in cellular recognition, e.g. in the case of inflammatory reactions, maturation of immune cells, differentiation processes, hormone-, pathogen- and toxin binding (Varki, A., Glycobiology, 2, pp. 25-40 (1992)).

Glycoconjugates containing Neu5Gc are immunogenic in humans. In healthy humans, Neu5Gc is not detectable, although Neu5Gc is abundant in most mammals. The lack of Neu5Gc in man is due to an exon deletion in the human gene that prevents the formation of functional enzyme (Chou, H. H., et al. Proc. Natl. Acad. Sci. (USA), 95, pp. 11751-11756 (1998); Irie, A., et al. J. Biol. Chem., 273, pp. 15866-15871 (1998)). Thus, Neu5Gc-containing glycoconjugates act as antigens and can induce the formation of antibodies. Historically, the antibodies have been referred to as Hanganutziu-Deicher (HD) antigens and antibodies (Hanganutziu, M., CR Soc. Biol. (Paris), 91, p. 1457 (1924); Deicher, H., Z. Hyg., 106, p. 561 (1926)). Hanganutziu-Deicher antigens are detectable in many human tumors (colon carcinoma, retinoblastoma, melanoma and carcinoma of the breast) as well as in chicken tumor tissues (Higashi, H., et al. Cancer Res., 45, pp. 3796-3802 (1985)). Although the amount of antigen in tumors is very small (usually less than 1% of the total amount of sialic acid, often in the range of from 0.01 to 0.1%), it is capable of inducing the formation of Hanganutziu-Deicher antibodies (Higashihara, T., et al., Int Arch Allergy Appl Immunol., 95, pp. 231-235 (1991)). This immunological reaction is a potential barrier to xenotransplantation of Neu5Gc-containing pig organs to humans.

The Neu5Gc epitope is formed by the addition of a hydroxyl group to the N-acetyl moiety of Neu5Ac. The enzyme that catalyzes the hydroxylation is CMP-Neu5Ac hydroxylase. Thus, the expression of the CMP-Neu5Ac hydroxylase gene determines the presence of the Neu5Gc epitope on cell surfaces. Purification studies of CMP-Neu5Ac hydroxylase in mammals have shown that it is a soluble, cytosolic oxygenase that is dependent on cytochrome b5 and cytochrome b5 reductase (Kawano, T., et al., J. Biol. Chem., 269, pp. 9024-9029 (1994); Schneckenburger, P., et al., Glycoconj. J., 11, pp. 194-203 (1994); Schlenzka, W., et al., Glycobiology, 4, pp. 675-683 (1994); Kozutsumi, Y., et al., J. Biochem. (Tokyo), 108, pp. 704-706 (1990); and, Shaw, L., et al. Eur. J. Biochem., 219, pp. 1001-1011 (1994)).

Another important feature of Neu5Gc is that it acts as an adhesion molecule for pathogens, allowing for entry into the cell (Kelm, S. and Schauer, R., Int. Rev. Cytol, 179, pp. 137-240 (1997)). This causes disease and economic losses in certain livestock species. Specifically, enterotoxigenic Escherichia coli with K99 fimbriae infect newborn piglets by binding to Neu5Gc in gangliosides such as Nue5Gcα2→3Galβ1→4Glcβ1→1′ ceramide [GM3(Neu5Gc)], N-glycolylsialoparagloboside and GM2(Neu5Gc) attached to intestinal absorptive and mucus secreting cells, causing a potentially lethal diarrhea (Malykh, Y., et. al., Biochem. J., 370, pp. 601-607 (2003); Kyogashima, M., et al., (1993); Teneberg, S., et al., FEBS Letters, 263, pp. 10-14 (1990); Isobe, T., et al., Anal. Biochem., 236, pp. 35-40 (1996); Lindahl, M. and Carlstedt, I., J. Gen. Microbiol., 136, pp. 1609-1614 (1990); King, T. P., et al., Proceedings of the 6^(th) International Symposium on Digestive Physiology in Pigs, pp. 290-293, (1994)). Pig rotavirus infects pig newborns causing diarrhea by binding to GM3(Neu5Gc). Pig transmissible gastroenteritis coronavirus infects pigs via entry into glycoconjugates containing α2,3-bound Neu5Gc (Schultz, B., et al., J. Virol., 70, pp. 5634-5637 (1996)).

CMP-Neu5Ac hydroxylase has been isolated from mouse liver and pig submandibular glands to homogeneity and characterized (Kawano, T., et al., J. Biol. Chem., 269, pp. 9024-9029 (1994); Schneckenburger, P., et al., Glycoconj. J., 11, pp. 194-203 (1994); and, Schlenzka, W., et al., Glycobiology, 4, pp. 675-683 (1994)).

Schlenzka, et al. (Glycobiology, Vol. 4, pp. 675-683 (1994)) purified the enzyme from pig submandibular glands using ion exchange chromatography, chromatography with immobilized triazin dyes, hydrophobic interaction chromatography and gel filtration. Schneckenburger et al. (Glycoconj. J., Vol. 11, pp. 194-203 (1994)) isolated the CMP-Neu5Ac hydroxylase from mouse liver. Both the CMP-Neu5Ac hydroxylase from pig submandibular glands and the one from mouse liver are soluble monomers having a molecular weight of 65 kDa. Their catalytic interactions with CMP-Neu5Ac and cytochrome b5 are very similar to one another. The activity of these enzymes seems to be dependent on an iron-containing prosthetic group.

JP-A 06 113838 describes the protein and DNA sequences of murine CMP-Neu5Ac hydroxylase, as well as a monoclonal antibody that specifically binds to the hydroxylase.

PCT Publication No. WO 97/03200A1 to Boehringer Manheim GMBH discloses a partial cDNA for the porcine CMP-Neu5Ac hydroxylase. This application discloses a cDNA sequence beginning in the middle of Exon 8 of the CMP-Neu5Ac hydroxylase gene (further disclosed as GenBank accession number Y15010).

Martensen, L., et al. (Eur. J. Biochem., Vol. 268, pp. 5157-5166 (2001)) discloses a full length amino acid sequence of porcine CMP-Neu5Ac hydroxylase.

PCT Publication No. WO 02/088351 to RBC Biotechnology discloses a partial cDNA and genomic sequence (exons 7-11 as well as partial genomic sequence surrounding each exon) of porcine CMP-NeuAc hydroxylase. In addition, methods are provided to generate porcine cells and animals lacking the CMP-NeuAc hydroxylase epitope, optionally, in combination with other genetic modifications, such as inactivation of the alpha-1,3-galactosyltransferase gene and/or insertion of complement proteins.

It is an object of the present invention to provide genomic and regulatory sequences of the porcine CMP-Neu5Ac hydroxylase gene.

It is an object of the present invention to provide the full length cDNA, as well as novel variants of the CMP-Neu5Ac hydroxylase gene.

It is another object of the invention to provide novel nucleic acid and amino acid sequences that encode the CMP-Neu5Ac hydroxylase gene.

It is yet a further object of the present invention to provide cells, tissues and/or organs deficient in the CMP-Neu5Ac hydroxylase gene.

It is another object of the present invention to generate animals, particularly pigs, lacking a functional CMP-Neu5Ac hydroxylase gene.

It is yet a further object of the present invention to provide cells, tissues and/or organs deficient in the CMP-Neu5Ac hydroxylase gene for use in xenotransplantation of non-human organs to human recipients in need thereof.

SUMMARY OF THE INVENTION

The full length cDNA sequence, peptide sequence, and genomic organization of the porcine CMP-Neu5Ac hydroxylase gene has been determined. To date, only partial cDNA and genomic sequences have been identified. The present invention provides novel porcine CMP-Neu5Ac hydroxylase protein, cDNA, cDNA variants, and genomic DNA sequence. Furthermore, the present invention includes porcine animals, tissues, and organs, as well as cells and cell lines derived from such animals, tissue, and organs, which lack expression of functional CMP-Neu5Ac hydroxylase. Such animals, tissues, organs, and cells can be used in research and in medical therapy, including xenotransplantation. In addition, methods are provided to prepare organs, tissues, and cells lacking the porcine CMP-Neu5Ac hydroxylase gene for use in xenotransplantation.

One aspect of the present invention provides the full length cDNA of porcine CMP-Neu5Ac hydroxylase. The full length cDNA is shown in Table 1 (SEQ ID No 1) and the full length peptide sequence is provided in Table 2 (SEQ ID No 2). The start codon for the full-length cDNA is located in the 3′ portion of Exon 4, and the stop codon is found in the 3′ portion of Exon 17. Nucleotide and amino acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to SEQ ID Nos 1 or 2 are provided. In addition, nucleotide and peptide sequences that contain at least 10, 15, 17, 20, 25 or 30 nucleotide or amino acid sequences of SEQ ID Nos 1 or 2 are also provided. Further provided is any nucleotide sequence that hybridizes, optionally under stringent conditions, to SEQ ID No 1, as well as, nucleotides homologous thereto.

In one embodiment, nucleic acid and peptide sequences encoding three novel variants of CMP-Neu5Ac hydroxylase are provided (Tables 3-8, FIG. 2). SEQ ID No 3 represents the cDNA of a variant of the gene, variant-1, that includes Exons 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15a, 16, 17, and 18. SEQ ID No 5 represents the cDNA of a variant of the gene, variant-2, that includes Exons 1, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 12a. SEQ ID No 7 represents the cDNA of a variant of the gene, variant-3, that includes Exons 1, 4, 5, 6, 7, 8, 9, 10, 11 and 11a. SEQ ID Nos 4, 6 and 8 represent the amino acid sequences of variant-1, variant-2 and variant-3, respectively. Nucleotide and amino acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to SEQ ID Nos 3-8 are provided. In addition, nucleotide and peptide sequences that contain at least 10, 15, 17, 20, 25, or 30 nucleotide or amino acid sequence of SEQ ID Nos 3-8 are also provided. Further provided is any nucleotide sequence that hybridizes, optionally under stringent conditions, to SEQ ID Nos 3, 5 and 7, as well as, nucleotides homologous thereto.

A further embodiment provides nucleic acid sequences representing genomic DNA sequences of the CMP-Neu5Ac hydroxylase gene (Table 9, FIG. 1). SEQ ID Nos 10-28 represent Exons 1, 4-11, 11a, 12, 12a, 13-15, 15a, 16-18, respectively, and SEQ ID Nos 29-45 represent Introns 1a, 1b, 4-15, 15a, 16, and 17, respectively. SEQ ID No. 9 represents the 5′ untranslated region of the CMP-Neu5Ac hydroxylase gene. SEQ ID No. 46 (Table 10) represents the genomic DNA and regulatory sequence of CMP-Neu5Ac hydroxylase.

In another embodiment, the genomic sequence of the porcine CMP-Neu5Ac hydroxylase gene is represented by SEQ ID No. 47. SEQ ID No. 47 represents the 5′ contiguous genomic sequence containing 5′ UTR, Exon 1 and a portion of intronic sequence located 3′ of Exon 1 (Table 11).

In another embodiment, the genomic sequence of the porcine CMP-Neu5Ac hydroxylase gene is represented by SEQ ID No. 48. SEQ ID NO. 48 represents a contiguous genomic sequence containing intronic sequence located 5′ to Exon 4, Exon 4, Intron 4, Exon 5, Intron 5, Exon 6, Intron 6, Exon 7, Intron 7, Exon 8, Intron 8, Exon 9, Intron 9, Exon 10, Intron 10, Exon 11, Intron 11, Exon 12, Intron 12, Exon 13, Intron 13, Exon 14, Intron 14, Exon 15, Intron 15, Exon 16, Intron 16, Exon 17, Intron 17, and Exon 18 (Table 12). In addition, nucleotide sequences that contain at least 2775, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 or 10,000 contiguous nucleotides of SEQ ID NO. 48 are provided, as well as nucleotide sequences at least 80, 85, 90, 95, 98, or 99% homologous to SEQ ID NO. 48.

In another embodiment, the genomic sequence of the porcine CMP-Neu5Ac hydroxylase gene is represented by SEQ ID No. 49. SEQ ID NO. 49 represents contiguous genomic sequences containing Intronic sequence 5′ to Exon 4, Exon 4, Intron 4, Exon 5, Intron 5, Exon 6, Intron 6, Exon 7, Intron 7 and Exon 8. Further, nucleotide sequences that contain at least 1750, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, or 20000 contiguous nucleotides of SEQ ID NO. 49 are provided, as well as nucleotide sequences at least 80, 85, 90, 95, 98, or 99% homologous to SEQ ID NO. 49.

In another embodiment, the genomic sequence of the porcine CMP-Neu5Ac hydroxylase gene is represented by SEQ ID No. 50. SEQ ID NO. 50 represents contiguous genomic sequences containing Exon 12, Intron 12, Exon 13, Intron 13, Exon 14, Intron 14, Exon 15, Intron 15, Exon 16, Intron 16, Exon 17, Intron 17, and Exon 18 are provided. Nucleotide sequences that contain at least 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000 or 20,000 contiguous nucleotides of SEQ ID NO. 50 are provided, as well as nucleotide sequences at least 80, 85, 90, 95, 98, or 99% homologous to SEQ ID NO. 50.

In further embodiments, nucleotide and amino acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to SEQ ID Nos 9-45, 46, 47, 48, 49 and 50 are provided. In addition, nucleotide and peptide sequences that contain at least 10, 15, 17, 20, 25, 30, 50, 100, 150, 200, 300, 400, 500 or 1000 contiguous nucleotide or amino acid sequences of SEQ ID Nos 9-45, 46, 47, and 48 are also provided. Further provided is any nucleotide sequence that hybridizes, optionally under stringent conditions, to SEQ ID Nos 9-45, 46, 47, 48, 49 and 50, as well as, nucleotides homologous thereto.

Another aspect of the present invention provides nucleic acid constructs that contain cDNA or variants thereof encoding CMP-Neu5Ac hydroxylase. These cDNA sequences can be derived from Seq ID Nos. 1-8, or any fragment thereof. Constructs can contain one, or more than one, internal ribosome entry site (IRES). The construct can also contain a promoter operably linked to the nucleic acid sequence encoding CMP-Neu5Ac hydroxylase, or, alternatively, the construct can be promoterless. In another embodiment, nucleic acid constructs are provided that contain nucleic acid sequences that permit random or targeted insertion into a host genome. In addition to the nucleic acid sequences the expression vector can contain selectable marker sequences, such as, for example, enhanced Green Fluorescent Protein (eGFP) gene sequences, initiation and/or enhancer sequences, poly A-tail sequences, and/or nucleic acid sequences that provide for the expression of the construct in prokaryotic and/or eukaryotic host cells.

In another embodiment, nucleic acid targeting vectors constructs are also provided wherein homologous recombination in somatic cells can be achieved. These targeting vectors can be transformed into mammalian cells to target the CMP-Neu5Ac hydroxylase gene via homologous recombination. In one embodiment, the targeting vectors can contain a 3′ recombination arm and a 5′ recombination arm that is homologous to the genomic sequence of a CMP-Neu5Ac hydroxylase. The homologous DNA sequence can include at least 15 bp, 20 bp, 25 bp, 50 bp, 100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp of sequence homologous to the CMP-Neu5Ac hydroxylase sequence. In another embodiment, the homologous DNA sequence can include one or more intron and/or exon sequences. In a specific embodiment, the DNA sequence can be homologous to Intron 5 and Intron 6 of the CMP-Neu5Ac hydroxylase gene (see, for example, FIGS. 6-8). In another specific embodiment, the DNA sequence can be homologous to Intron 5, a 55 bp portion of Exon 6, and Intron 6 of the CMP-Neu5Ac hydroxylase gene, and contain enhanced Green Fluorescent Protein sequence in an in-frame orientation 3′ to the 55 bp portion of Exon 6 (see, for example, FIGS. 10 and 11).

Another embodiment of the present invention provides oligonucleotide primers capable of hybridizing to porcine CMP-Neu5Ac hydroxylase cDNA or genomic sequence, such as Seq ID Nos. 1, 3, 5, 7, 9-45, 46, 47 or 48. In a preferred embodiment, the primers hybridize under stringent conditions to SEQ ID Nos. 1, 3, 5, 7, 9-45, 46, 47 or 48. Another embodiment provides oligonucleotide probes capable of hybridizing to porcine CMP-Neu5Ac hydroxylase nucleic acid sequences, such as SEQ ID Nos. 1, 3, 5, 7, 9-45, 46, 47, or 48. The polynucleotide primers or probes can have at least 14 bases, 20 bases, preferably 30 bases, or 50 bases which hybridize to a polynucleotide of the present invention. The probe or primer can be at least 14 nucleotides in length, and in a preferred embodiment, are at least 15, 20, 25, 28, or 30 nucleotides in length.

In another aspect of the present invention, mammalian cells lacking at least one allele of the CMP-Neu5Ac hydroxylase gene produced according to the process, sequences and/or constructs described herein are provided. These cells can be obtained as a result of homologous recombination. Particularly, by inactivating at least one allele of the CMP-NeuAc hydroxylase gene, cells can be produced which have reduced capability for expression of functional Hanganutziu-Deicher antigens.

In embodiments of the present invention, alleles of the CMP-Neu5Ac hydroxylase gene are rendered inactive according to the process, sequences and/or constructs described herein, such that the resultant CMP-Neu5Ac hydroxylase enzyme can no longer generate Hanganutziu-Deicher antigens. In one embodiment, the CMP-Neu5Ac hydroxylase gene can be transcribed into RNA, but not translated into protein. In another embodiment, the CMP-Neu5Ac hydroxylase gene can be transcribed in an inactive truncated form. Such a truncated RNA may either not be translated or can be translated into a nonfunctional protein. In an alternative embodiment, the CMP-Neu5Ac hydroxylase gene can be inactivated in such a way that no transcription of the gene occurs. In a further embodiment, the CMP-Neu5Ac hydroxylase gene can be transcribed and then translated into a nonfunctional protein.

In a further aspect of the present invention, porcine animals are provided in which at least one allele of the CMP-Neu5Ac hydroxylase gene is inactivated via a genetic targeting event produced according to the process, sequences and/or constructs described herein. In another aspect of the present invention, porcine animals are provided in which both alleles of the CMP-Neu5Ac hydroxylase gene are inactivated via a genetic targeting event. The gene can be targeted via homologous recombination. In other embodiments, the gene can be disrupted, i.e. a portion of the genetic code can be altered, thereby affecting transcription and/or translation of that segment of the gene. For example, disruption of a gene can occur through substitution, deletion (“knock-out”) or insertion (“knock-in”) techniques. Additional genes for a desired protein or regulatory sequence that modulate transcription of an existing sequence can be inserted.

In another aspect of the present invention, porcine cells lacking one allele, optionally both alleles of the porcine CMP-Neu5Ac hydroxylase gene can be used as donor cells for nuclear transfer into enucleated oocytes to produce cloned, transgenic animals. Alternatively, porcine CMP-Neu5Ac hydroxylase knockouts can be created in embryonic stem cells, which are then used to produce offspring. Offspring lacking a single allele of the functional CMP-Neu5Ac hydroxylase gene produced according to the process, sequences and/or constructs described herein can be breed to further produce offspring lacking functionality in both alleles through mendelian type inheritance. Cells, tissues and/or organs can be harvested from these animals for use in xenotransplantation strategies. The elimination of the Hanganutziu-Deicher antigens can reduce the immune rejection of the transplanted cell, tissue or organ due to the Neu5Gc epitope.

Alternatively, animals lacking at least one allele of the CMP-Neu5Ac hydroxylase gene produced according to the process, sequences and/or constructs described herein can be less susceptible or resistant to enterotoxigenic infection and disease such as, for example, E. Coli infection, rotavirus infection, and gastroenteritis coronavirus. Such animals can be used, for example, in commercial farming.

In one aspect of the present invention, a pig can be prepared by a method in accordance with any aspect of the present invention. Genetically modified pigs can be used as a source of tissue and/or organs for transplantation therapy. A pig embryo prepared in this manner or a cell line developed therefrom can also be used in cell-transplantation therapy. Accordingly, there is provided in a further aspect of the invention a method of therapy comprising the administration of genetically modified cells lacking porcine CMP-Neu5Ac hydroxylase to a patient, wherein the cells have been prepared from an embryo or animal lacking CMP-Neu5Ac hydroxylase. This aspect of the invention extends to the use of such cells in medicine, e.g. cell-transplantation therapy, and also to the use of cells derived from such embryos in the preparation of a cell or tissue graft for transplantation. The cells can be organized into tissues or organs, for example, heart, lung, liver, kidney, pancreas, corneas, nervous (e.g. brain, central nervous system, spinal cord), skin, or the cells can be islet cells, blood cells (e.g. haemocytes, i.e. red blood cells, leucocytes) or haematopoietic stem cells or other stem cells (e.g. bone marrow).

In another aspect of the present invention, CMP-Neu5Ac hydroxylase-deficient pigs also lack genes encoding other xenoantigens, such as, for example, porcine iGb3 synthase (see, for example, U.S. Patent Application 60/517,524), and/or porcine Forssman synthase (see, for example, U.S. Patent Application 60/568,922). In another embodiment, porcine cells are provided that lack the α1,3 galactosyltransferase gene and the CMP-Neu5Ac hydroxylase gene produced according to the process, sequences and/or constructs described herein. In another embodiment, porcine α1,3 galactosyltransferase gene knockout cells are further modified to knockout the CMP-Neu5Ac hydroxylase gene produced according to the process, sequences and/or constructs described herein. In addition, CMP-Neu5Ac hydroxylase deficient pigs produced according to the process, sequences and/or constructs described herein, optionally lacking one or more additional genes associated with an adverse immune response, can be modified to express complement inhibiting proteins, such as, for example, CD59, DAF, and/or MCP can be further modified to eliminate the expression of al least one allele of the CMP-Neu5Ac hydroxylase gene. These animals can be used as a source of tissue and/or organs for transplantation therapy. These animals can be used as a source of tissue and/or organs for transplantation therapy. A pig embryo prepared in this manner or a cell line developed therefrom can also be used in cell-transplantation therapy.

DESCRIPTION OF THE INVENTION

Elimination of the CMP-Neu5Ac hydroxylase gene produced according to the process, sequences and/or constructs described herein can reduce a human beings immunological response to the Neu5Gc epitope and remove an immunological barrier to xenotransplantation. The present invention is directed to novel nucleic acid sequences encoding the full-length cDNA and peptide. Information about the genomic organization, intronic sequences and regulatory regions of the gene are also provided. In one aspect, the invention provides isolated and substantially purified cDNA molecules having one of SEQ ID Nos: 1, 3, 5 or 7, or a fragment thereof. In another aspect of the invention, DNA sequences comprising the full-length genome of the CMP-NeuAc hydrolase gene are provided in SEQ ID Nos 9-45, 46, 47, 48, 49 or 50 or fragments thereof. In another aspect, primers for amplifying porcine CMP-Neu5Ac hydroxylase cDNA or genomic sequence derived from SEQ ID Nos. 1, 3, 5, 7, 9-45, 46, 47, 48, 49 or 50 are provided. Additionally probes for identifying CMP-Neu5Ac hydroxylase nucleic acid sequences derived from SEQ ID Nos. 1, 3, 5, 7, 9-45, 46, 47, 48, 49 or 50, or fragments thereof are provided. DNA represented by SEQ ID Nos 9-45, 46, 47, 48, 49 or 50, or fragments thereof, can be used to construct pigs lacking functional CMP-Neu5Ac hydroxylase genes. Thus, the invention also provides a porcine chromosome lacking a functional CMP-NeuAc hydroxylase gene and a transgenic pig lacking a functional CMP-NeuAc hydroxylase protein produced according to the process, sequences and/or constructs described herein. Such pigs can be used as tissue sources for xenotransplantation into humans. In an alternate embodiment, CMP-NeuAc hydroxylase-deficient pigs produced according to the process, sequences and/or constructs described herein also lack other genes associated with adverse immune responses in xenotransplantation, such as, for example, the α1,3 galactosyltransferase gene, iGb3 synthetase gene, or FSM synthase gene. In another embodiment, pigs lacking CMP-Neu5Ac hydroxylase produced according to the process, sequences and/or constructs described herein and/or other genes associated with adverse immune responses in xenotransplantation express complement inhibiting factors such as, for example, CD59, DAF, and/or MCP.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents the genomic organization of the porcine CMP-Neu5Ac hydroxylase gene. Closed bars depict each numbered exon. The length of the introns between the exons illustrates relative distances. (Open boxes also represent exons that appear in some variants (see FIG. 2); “start” and “stop” denote start and stop codons, respectively) The approximate scale is depicted in the bottom of the figure.

FIG. 2 depicts cDNA sequences of the CMP-Neu5Ac hydroxylase gene. Variant-1 contains exon 15a in place of exons 14 and 15. Variant-2 contains exon 12a, and variant-3 contains exon 11a. “Start” and “stop” denote the start and stop codons, respectively.

FIG. 3 illustrates four non-limiting examples of targeting vectors, along with their corresponding genomic organization. The selectable marker gene in this particular non-limiting example is eGFP (enhanced green fluorescent protein). eGFP can be inserted in the DNA constructs to inactivate the porcine CMP-NeuAc hydroxylase gene.

FIG. 4 illustrates transcription factor binding sites located within exon 1 (228 bp) and its 5′-flanking region spanning 601 bp.

FIG. 5 depicts oligonucleotide sequences that can be used for DNA construction of porcine CMP-Neu5Ac hydroxylase gene targeting vector.

FIG. 6 is a schematic diagram illustrating the production of a 3′-arm segment from the porcine CMP-Neu5Ac hydroxylase gene using primers pDH3 and pDH4, and its insertion into a vector (pCRII).

FIG. 7 is a schematic diagram illustrating the production of a 5′-arm segment from the porcine CMP-Neu5Ac hydroxylase gene using primers pDH1 and pDH2, followed by pDH2a, pDH2b, and pDH2c, and its insertion into a vector (pCRII) in which a 3′-arm has previously been inserted.

FIG. 8 is a non-limiting example of a schematic illustrating a targeting vector that can be utilized to delete Exon 6 of the porcine CMP-Neu5Ac hydroxylase gene through homologous recombination.

FIG. 9 represents oligonucleotide sequences used in generating a enhanced green fluorescent protein expression vector for use in a Knock-in strategy.

FIG. 10 is a schematic illustrating the insertion of a EGFP fragment with a polyA signal into the targeting vector pDHΔex6.

FIG. 11 is a schematic illustrating a knock-in vector for expression of eGFP.

FIG. 12 is a schematic illustrating homologous recombination resulting in a frameshift between the targeting cassette DNA construct (pDHΔex6) and genomic DNA.

FIG. 13 is a schematic illustrating homologous recombination resulting in a frameshift between the targeting cassette DNA construct (pDHΔex6) and genomic DNA.

DEFINITIONS

A “target DNA sequence” is a DNA sequence to be modified by homologous recombination. The target DNA can be in any organelle of the animal cell including the nucleus and mitochondria and can be an intact gene, an exon or intron, a regulatory sequence or any region between genes.

A “targeting DNA sequence” is a DNA sequence containing the desired sequence modifications. The targeting DNA sequence can be substantially isogenic with the target DNA.

A “homologous DNA sequence or homologous DNA” is a DNA sequence that is at least about 80%, 85%, 90%, 95%, 98% or 99% identical with a reference DNA sequence. A homologous sequence hybridizes under stringent conditions to the target sequence, stringent hybridization conditions include those that will allow hybridization occur if there is at least 85% and preferably at least 95% or 98% identity between the sequences.

An “isogenic or substantially isogenic DNA sequence” is a DNA sequence that is identical to or nearly identical to a reference DNA sequence. The term “substantially isogenic” refers to DNA that is at least about 97-99% identical with the reference DNA sequence, and preferably at least about 99.5-99.9% identical with the reference DNA sequence, and in certain uses 100% identical with the reference DNA sequence.

“Homologous recombination” refers to the process of DNA recombination based on sequence homology.

“Gene targeting” refers to homologous recombination between two DNA sequences, one of which is located on a chromosome and the other of which is not.

“Non-homologous or random integration” refers to any process by which DNA is integrated into the genome that does not involve homologous recombination.

A “selectable marker gene” is a gene, the expression of which allows cells containing the gene to be identified. A selectable marker can be one that allows a cell to proliferate on a medium that prevents or slows the growth of cells without the gene. Examples include antibiotic resistance genes and genes which allow an organism to grow on a selected metabolite. Alternatively, the gene can facilitate visual screening of transformants by conferring on cells a phenotype that is easily identified. Such an identifiable phenotype can be, for example, the production of luminescence or the production of a colored compound, or the production of a detectable change in the medium surrounding the cell.

The term “contiguous” is used herein in its standard meaning, i.e., without interruption, or uninterrupted.

The term “porcine” refers to any pig species, including pig species such as Large White, Landrace, Meishan, Minipig.

The term “oocyte” describes the mature animal ovum which is the final product of oogenesis and also the precursor forms being the oogonium, the primary oocyte and the secondary oocyte respectively.

The term “fragment” means a portion or partial sequence of a nucleotide or peptide sequence.

The terms “derivative” and “analog” means a nucleotide or peptide sequence which retains essentially the same biological function or activity as such nucleotide or peptide. For example, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

DNA (deoxyribonucleic acid) sequences provided herein are represented by the bases adenine (A), thymine (T), cytosine (C), and guanine (G).

Amino acid sequences provided herein are represented by the following abbreviations:

A alanine P proline B aspartate or asparagine Q glutamine C cysteine R arginine D aspartate S serine E glutamate T threonine F phenylalanine G glycine V valine H histidine W tryptophan I isoleucine Y tyrosine Z glutamate or glutamine K lysine L leucine M methionine N asparagine

“Transfection” refers to the introduction of DNA into a host cell. Cells do not naturally take up DNA. Thus, a variety of technical “tricks” are utilized to facilitate gene transfer. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaPO₄ and electroporation. (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, 1989). Transformation of the host cell is the indicia of successful transfection.

I. Complete cDNA Sequence and Variants of the Porcine CMP-Neu5Ac Hydroxylase Gene

One aspect of the present invention provides novel, full length nucleic acid cDNA sequences of the porcine CMP-Neu5Ac hydroxylase gene (FIG. 2, Table 1, Seq ID No 1). Another aspect of the present invention provides predicted amino acid peptide sequences of the porcine CMP-Neu5Ac hydroxylase gene (Table 2, Seq ID No 2). The ATG start codon for the full-length cDNA is located in the 3′ portion of Exon 4, and the stop codon TAG is found in the 3′ portion of Exon 17. Nucleic and amino acid sequences at least 90, 95, 98 or 99% homologous to Seq ID Nos 1 or 2 are provided. In addition, nucleotide and peptide sequences that contain at least 10, 15, 17, 20 or 25 contiguous nucleic or amino acids of Seq ID Nos 1 or 2 are also provided. Further provided are fragments, derivatives and analogs of Seq ID Nos 1-2. Fragments of Seq ID Nos. 1-2 can include any contiguous nucleic acid or peptide sequence that includes at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90. 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 700, 750, 800, 850, 900, 1000, 5000 or 10,000 nucleotides.

TABLE 1 Full length cDNA CCCGACGTCCTGGCAGCGCCCAGGCACTGTTA Exons Seq ID No 1 TTGGTGCCTCCTGTGTCCACGCGCTTCCCGGC 1 & CAGGCAGCCCTGGCGGATCCTATTTTCTGTTC 4-18 CCCCGATTCTGGTACCTCTCCCTCCCGCCCTC GGTGCGCAGCCGTCCTCCTGCAGTGCCTGCTC CTCCAGGGGCGAAACCGATCAGGGATCAGGCC ACCCGCCTCCTGAACATCCCTCCTTAGTTCCC ACAGTCTAATGCCTTGTGGAAGCAAATGAGCC ACAGAAGCTGAAGGAAAAACCACCATTCTTTC TTAATACCTGGAGAGAGGCAACGACAGACTAT GAGCAGCATCGAACAAACGACGGAGATCCTGT TGTGCCTCTCACCTGCCGAAGCTGCCAATCTC AAGGAAGGAATCAATTTTGTTCGAAATAAGAG CACTGGCAAGGATTACATCTTATTTAAGAATA AGAGCCGCCTGAAGGCATGTAAGAACATGTGC AAGCACCAAGGAGGCCTCTTCATTAAAGACAT TGAGGATCTAAATGGAAGGTCTGTTAAATGCA CAAAACACAACTGGAAGTTAGATGTAAGCAGC ATGAAGTATATCAATCCTCCTGGAAGCTTCTG TCAAGACGAACTGGTTGTAGAAAAGGATGAAG AAAATGGAGTTTTGCTTCTAGAACTAAATCCT CCTAACCCGTGGGATTCAGAACCCAGATCTCC TGAAGATTTGGCATTTGGGGAAGTGCAGATCA CGTACCTTACTCACGCCTGCATGGACCTCAAG CTGGGGGACAAGAGAATGGTGTTCGACCCTTG GTTAATCGGTCCTGCTTTTGCGCGAGGATGGT GGTTACTACACGAGCCTCCATCTGATTGGCTG GAGAGGCTGAGCCGCGCAGACTTAATTTACAT CAGTCACATGCACTCAGACCACCTGAGTTACC CAACACTGAAGAAGCTTGCTGAGAGAAGACCA GATGTTCCCATTTATGTTGGCAACACGGAAAG ACCTGTATTTTGGAATCTGAATCAGAGTGGCG TCCAGTTGACTAATATCAATGTAGTGCCATTT GGAATATGGCAGCAGGTAGACAAAAATCTTCG ATTCATGATCTTGATGGATGGCGTTCATCCTG AGATGGACACTTGCATTATTGTGGAATACAAA GGTCATAAAATACTCAATACAGTGGATTGCAC CAGACCCAATGGAGGAAGGCTGCCTATGAAGG TTGCATTAATGATGAGTGATTTTGCTGGAGGA GCTTCAGGCTTTCCAATGACTTTCAGTGGTGG AAAATTTACTGAGGAATGGAAAGCCCAATTCA TTAAAACAGAAAGGAAGAAACTCCTGAACTAC AAGGCTCGGCTGGTGAAGGACCTACAACCCAG AATTTACTGCCCCTTTCCTGGGTATTTCGTGG AATCCCACCCAGCAGACAAGTATATTAAGGAA ACAAACATCAAAAATGACCCAAATGAACTCAA CAATCTTATCAAGAAGAATTCTGAGGTGGTAA CCTGGACCCCAAGACCTGGAGCCACTCTTGAT CTGGGTAGGATGCTAAAGGACCCAACAGACAG CAAGGGCATCGTAGAGCCTCCAGAAGGGACTA AGATTTACAAGGATTCCTGGGATTTTGGCCCA TATTTGAATATCTTGAATGCTGCTATAGGAGA TGAAATATTTCGTCACTCATCCTGGATAAAAG AATACTTCACTTGGGCTGGATTTAAGGATTAT AACCTGGTGGTCAGGATGATTGAGACAGATGA GGACTTCAGCCCTTTGCCTGGAGGATATGACT ATTTGGTTGACTTTCTGGATTTATCCTTTCCA AAAGAAAGACCAAGCCGGGAACATCCATATGA GGAAATTCGGAGCCGGGTTGATGTCATCAGAC ACGTGGTAAAGAATGGTCTGCTCTGGGATGAC TTGTACATAGGATTCCAAACCCGGCTTCAGCG GGATCCTGATATATACCATCATCTGTTTTGGA ATCATTTTCAAATAAAACTCCCCCTCACACCA CCTGACTGGAAGTCCTTCCTGATGTGCTCTGG GTAGAGAGGACCTGAGCTGTCCCAGGGGTGCC CAACAACATGAAAAAATCAAGAATTTATTGCT GCTACGTCAAAGCTTATACCAGAGATTATGCC TTATAGACATTAGCAATGGATAATTATATGTT GCACTTGTGAAATGTGCACATATCCTGTTTAT GAATCACCACATAGCCAGATTATCAATATTTT ACTTATTTCGTAAAAAATCCACAATTTTCCAT AACAGAATCAACGTGTGCAATAGGAACAAGAT TGCTATGGAAAACGAGGGTAACAGGAGGAGAT ATTAATCCAAGCATAGAAGAAATAGACAAATG AGGGGCCATAAGGGGAATATAGGGAAGAGAAA AAAATTAAGATGGAATTTTAAAAGGAGAATGT AAAAAATAGATATTTGTTCCTTAATAGGTTGA TTCCTCAAATAGAGCCCATGAATATAATCAAA TAGGAAGGGTTCATGACTGTTTTCAATTTTTC AAAAAGCTTTGTTGAAATCATAGACTTGCAAA ACAAGGCTGTAGAGGCCACCCTAAAATGGAAA ATTTCACTGGGACTGAAATTATTTTGATTCAA TGACAAAATTTGTTATTTACTGCGGATTATAA ACTCTAACAAATAGCGATCTCTTTGCTTCATA AAAACATAAACACTAGCTAGTAATAAAATGAG TTCTGCAG

TABLE 2 Full length Amino Acid Sequence M S S I E Q T T E I L L C L S P A E A A Seq ID No 2 N L K E G I N F V R N K S T G K D Y I L F K N K S R L K A C K N M C K H Q G G L F I K D I E D L N G R S V K C T K H N W K L D V S S M K Y I N P P G S F C Q D E L V V E K D E E N G V L L L E L N P P N P W D S E P R S P E D L A F G E V Q I T Y L T H A C M D L K L G D K R M V F D P W L I G P A F A R G W W L L H E P P S D W L E R L S R A D L I Y I S H M H S D H L S Y P T L K K L A E R R P D V P I Y V G N T E R P V F W N L N Q S G V Q L T N I N V V P F G I W Q Q V D K N L R F M I L M D G V H P E M D T C I I V E Y K G H K I L N T V D C T R P N G G R L P M K V A L M M S D F A G G A S G F P M T F S G G K F T E E W K A Q F I K T E R K K L L N Y K A R L V K D L Q P R I Y C P F P G Y F V E S H P A D K Y I K E T N I K N D P N E L N N L I K K N S E V V T W T P R P G A T L D L G R M L K D P T D S K G I V E P P E G T K I Y K D S W D F G P Y L N I L N A A I G D E I F R H S S W I K E Y F T W A G F K D Y N L V V R M I E T D E D F S P L P G G Y D Y L V D F L D L S F P K E R P S R E H P Y E E I R S R V D V I R H V V K N G L L W D D L Y I G F Q T R L Q R D P D I Y H H L F W N H F Q I K L P L T P P D W K S F L M C S G

Variants

Another aspect of the present invention provides novel nucleic acid cDNA sequences of three novel variants of CMP-Neu5Ac hydroxylase gene transcript (FIG. 2, Tables 3, 5, and 7, Seq ID Nos. 3, 5, and 7). Seq ID No 3 represents the cDNA of a variant of the gene, variant-1, that includes Exons 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15a, 16, 17, and 18. Exon 15a is a cryptic Exon that normally appears in Intron 15, approximately 460 bp upstream of Exon 16. The start codon for variant-1 is located in Exon 4, while the stop codon is located in Exon 17. Seq ID No 5 represents the cDNA of a variant of the gene, variant-2, that includes Exons 1, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 12a. Exon 12a is a cryptic Exon which is retained from a partial sequence of Intron 12 (see SEQ ID. No. 21). The start codon for variant-2 is located in Exon 4, while the stop codon is located in the terminal end of Exon 12a. Seq ID No 7 represents the cDNA of a variant of the gene, variant-3, that includes Exons 1, 4, 5, 6, 7, 8, 9, 10, 11 and 11a. Exon 11a is a cryptic Exon which is retained from a partial sequence of Intron 11 (see Seq ID No. 19). The start codon for variant-3 is located in Exon 4, while the stop codon is located in Exon 11a. Another aspect of the present invention provides predicted amino acid peptide sequences of three novel variants of the porcine CMP-Neu5Ac Hydroxylase gene transcript. Seq ID Nos 4, 6 and 8 represent the amino acid sequences of variant-1, variant-2 and variant-3, respectively. Nucleotide and amino acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 3-8 are provided. In addition, nucleotide and peptide sequences that contain at least 10, 15, 17, 20, 25, 30, 50, 100, 150, 200, 300, 400, 500 or 1000 contiguous nucleotide or amino acid sequences of Seq ID Nos 3-8 are also provided. Further provided are fragments, derivatives and analogs of Seq ID Nos 3-8. Fragments of Seq ID Nos. 3-8 can include any contiguous nucleic acid or peptide sequence that includes at least about 10 bp, 15 bp, 17 bp, 20 bp, 50 bp, 100 bp, 500 bp, 1 kbp, 5 kbp or 10 kbp.

TABLE 3 Variant-1 cDNA CCCGACGTCCTGGCAGCGCCCAGGCACTGTT Exons 1, Seq ID No 3 ATTGGTGCCTCCTGTGTCCACGCGCTTCCCG 4-13, GCCAGGCAGCCCTGGCGGATCCTATTTTCTG 15a, 16, TTCCCCCGATTCTGGTACCTCTCCCTCCCGC 17, 18 CCTCGGTGCGCAGCCGTCCTCCTGCAGTGCC TGCTCCTCCAGGGGCGAAACCGATCAGGGAT CAGGCCACCCGCCTCCTGAACATCCCTCCTT AGTTCCCACAGTCTAATGCCTTGTGGAAGCA AATGAGCCACAGAAGCTGAAGGAAAAACCAC CATTTCTTTCTTAATACCTGGAGAGAGGCAA CGACAGACTATGAGCAGCATCGAACAAACGA CGGAGATCCTGTTGTGCCTCTCACCTGCCGA AGCTGCCAATCTCAAGGAAGGAATCAATTTT GTTCGAAATAAGAGCACTGGCAAGGATTACA TCTTATTTAAGAATAAGAGCCGCCTGAAGGC ATGTAAGAACATGTGCAAGCACCAAGGAGGC CTCTTCATTAAAGACATTGAGGATCTAAATG GAAGGTCTGTTAAATGCACAAAACACAACTG GAAGTTAGATGTAAGCAGCATGAAGTATATC AATCCTCCTGGAAGCTTCTGTCAAGACGAAC TGGTTGTAGAAAAGGATGAAGAAAATGGAGT TTTGCTTCTAGAACTAAATCCTCCTAACCCG TGGGATTCAGAACCCAGATCTCCTGAAGATT TGGCATTTGGGGAAGTGCAGATCACGTACCT TACTCACGCCTGCATGGACCTCAAGCTGGGG GACAAGAGAATGGTGTTCGACCCTTGGTTAA TCGGTCCTGCTTTTGCGCGAGGATGGTGGTT ACTACACGAGCCTCCATCTGATTGGCTGGAG AGGCTGAGCCGCGCAGACTTAATTTACATCA GTCACATGCACTCAGACCACCTGAGTTACCC AACACTGAAGAAGCTTGCTGAGAGAAGACCA GATGTTCCCATTTATGTTGGCAACACGGAAA GACCTGTATTTTGGAATCTGAATCAGAGTGG CGTCCAGTTGACTAATATCAATGTAGTGCCA TTTGGAATATGGCAGCAGGTAGACAAAAATC TTCGATTCATGATCTTGATGGATGGCGTTCA TCCTGAGATGGACACTTGCATTATTGTGGAA TACAAAGGTCATAAAATACTCAATACAGTGG ATTGCACCAGACCCAATGGAGGAAGGCTGCC TATGAAGGTTGCATTAATGATGAGTGATTTT GCTGGAGGAGCTTCAGGCTTTCCAATGACTT TCAGTGGTGGAAAATTTACTGAGGAATGGAA AGCCCAATTCATTAAAACAGAAAGGAAGAAA CTCCTGAACTACAAGGCTCGGCTGGTGAAGG ACCTACAACCCAGAATTTACTGCCCCTTTCC TGGGTATTTCGTGGAATCCCACCCAGCAGAC AAGTATATTAAGGAAACAAACATCAAAAATG ACCCAAATGAACTCAACAATCTTATCAAGAA GAATTCTGAGGTGGTAACCTGGACCCCAAGA CCTGGAGCCACTCTTGATCTGGGTAGGATGC TAAAGGACCCAACAGACAGATCCTGTGTCAG GAGTTGGGATTCTTTGAAGATTCGGAGCCGG GTTGATGTCATCAGACACGTGGTAAAGAATG GTCTGCTCTGGGATGACTTGTACATAGGATT CCAAACCCGGCTTCAGCGGGATCCTGATATA TACCATCATCTGTTTTGGAATCATTTTCAAA TAAAACTCCCCCTCACACCACCTGACTGGAA GTCCTTCCTGATGTGCTCTGGGTAGAGAGGA CCTGAGCTGTCCCAGGGGTGCCCAACAACAT GAAAAAATCAAGAATTTATTGCTGCTACGTC AAAGCTTATACCAGAGATTATGCCTTATAGA CATTAGCAATGGATAATTATATGTTGCACTT GTGAAATGTGCACATATCCTGTTTATGAATC ACCACATAGCCAGATTATCAATATTTTACTT ATTTCGTAAAAAATCCACAATTTTCCATAAC AGAATCAACGTGTGCAATAGGAACAAGATTG CTATGGAAAACGAGGGTAACAGGAGGAGATA TTAATCCAAGCATAGAAGAAATAGACAAATG AGGGGCCATAAGGGGAATATAGGGAAGAGAA AAAAATTAAGATGGAATTTTAAAAGGAGAAT GTAAAAAATAGATATTTGTTCCTTAATAGGT TGATTCCTCAAATAGAGCCCATGAATATAAT CAAATAGGAAGGGTTCATGACTGTTTTCAAT TTTTCAAAAAGCTTTGTTGAAATCATAGACT TGCAAAACAAGGCTGTAGAGGCCACCCTAAA ATGGAAAATTTCACTGGGACTGAAATTATTT TGATTCAATGACAAAATTTGTTATTTACTGC GGATTATAAACTCTAACAAATAGCGATCTCT TTGCTTCATAAAAACATAAACACTAGCTAGT AATAAAATGAGTTCTGCAG

TABLE 4 Variant-1 Amino Acid Sequence M S S I E Q T T E I L L C L S P A E A A Seq ID No 4 N L K E G I N F V R N K S T G K D Y I L F K N K S R L K A C K N M C K H Q G G L F I K D I E D L N G R S V K C T K H N W K L D V S S M K Y I N P P G S F C Q D E L V V E K D E E N G V L L L E L N P P N P W D S E P R S P E D L A F G E V Q I T Y L T H A C M D L K L G D K R M V F D P W L I G P A F A R G W W L L H E P P S D W L E R L S R A D L I Y I S H M H S D H L S Y P T L K K L A E R R P D V P I Y V G N T E R P V F W N L N Q S G V Q L T N I N V V P F G I W Q Q V D K N L R F M I L M D G V H P E M D T C I I V E Y K G H K I L N T V D C T R P N G G R L P M K V A L M M S D F A G G A S G F P M T F S G G K F T E E W K A Q F I K T E R K K L L N Y K A R L V K D L Q P R I Y C P F P G Y F V E S H P A D K Y I K E T N I K N D P N E L N N L I K K N S E V V T W T P R P G A T L D L G R M L K D P T D R S C V R S W D S L K I R S R V D V I R H V V K N G L L W D D L Y I G F Q T R L Q R D P D I Y H H L F W N H F Q I K L P L T P P D W K S F L M C S G

TABLE 5 Variant-2 cDNA CCCGACGTCCTGGCAGCGCCCAGGCACTG Exons 1, Seq ID No 5 TTATTGGTGCCTCCTGTGTCCACGCGCTT 4-12, 12a CCCGGCCAGGCAGCCCTGGCGGATCCTAT TTTCTGTTCCCCCGATTCTGGTACCTCTC CCTCCCGCCCTCGGTGCGCAGCCGTCCTC CTGCAGTGCCTGCTCCTCCAGGGGCGAAA CCGATCAGGGATCAGGCCACCCGCCTCCT GAACATCCCTCCTTAGTTCCCACAGTCTA ATGCCTTGTGGAAGCAAATGAGCCACAGA AGCTGAAGGAAAAACCACCATTCTTTCTT AATACCTGGAGAGAGGCAACGACAGACTA TGAGCAGCATCGAACAAACGACGGAGATC CTGTTGTGCCTCTCACCTGCCGAAGCTGC CAATCTCAAGGAAGGAATCAATTTTGTTC GAAATAAGAGCACTGGCAAGGATTACATC TTATTTAAGAATAAGAGCCGCCTGAAGGC ATGTAAGAACATGTGCAAGCACCAAGGAG GCCTCTTCATTAAAGACATTGAGGATCTA AATGGAAGGTCTGTTAAATGCACAAAACA CAACTGGAAGTTAGATGTAAGCAGCATGA AGTATATCAATCCTCCTGGAAGCTTCTGT CAAGACGAACTGGTTGTAGAAAAGGATGA AGAAAATGGAGTTTTGCTTCTAGAACTAA ATCCTCCTAACCCGTGGGATTCAGAACCC AGATCTCCTGAAGATTTGGCATTTGGGGA AGTGCAGATCACGTACCTTACTCACGCCT GCATGGACCTCAAGCTGGGGGACAAGAGA ATGGTGTTCGACCCTTGGTTAATCGGTCC TGCTTTTGCGCGAGGATGGTGGTTACTAC ACGAGCCTCCATCTGATTGGCTGGAGAGG CTGAGCCGCGCAGACTTAATTTACATCAG TCACATGCACTCAGACCACCTGAGTTACC CAACACTGAAGAAGCTTGCTGAGAGAAGA CCAGATGTTCCCATTTATGTTGGCAACAC GGAAAGACCTGTATTTTGGAATCTGAATC AGAGTGGCGTCCAGTTGACTAATATCAAT GTAGTGCCATTTGGAATATGGCAGCAGGT AGACAAAAATCTTCGATTCATGATCTTGA TGGATGGCGTTCATCCTGAGATGGACACT TGCATTATTGTGGAATACAAAGGTCATAA AATACTCAATACAGTGGATTGCACCAGAC CCAATGGAGGAAGGCTGCCTATGAAGGTT GCATTAATGATGAGTGATTTTGCTGGAGG AGCTTCAGGCTTTCCAATGACTTTCAGTG GTGGAAAATTTACTGAGGAATGGAAAGCC CAATTCATTAAAACAGAAAGGAAGAAACT CCTGAACTACAAGGCTCGGCTGGTGAAGG ACCTACAACCCAGAATTTACTGCCCCTTT CCTGGGTATTTCGTGGAATCCCACCCAGC AGACAAGTATGGCTGGATATTTTATATAA CGTGTTTACGCATAAGTTAATATATGCTG AATGAGTGATTTAGCTGTGAAACAACATG AAATGAGAAAGAATGATTAGTAGGGGTCT GGAGCTTATTTTAACAAGCAGCCTGAAAA CAGAAAGTATGAATAAAAAAAATTAAATG CAAAAAAAAAAAAAAAAAAAAAAAAAAAA

TABLE 6 Variant-2 Amino Acid Sequence M S S I E Q T T E I L L C L S P A E A A Seq ID No 6 N L K E G I N F V R N K S T G K D Y I L F K N K S R L K A C K N M C K H Q G G L F I K D I E D L N G R S V K C T K H N W K L D V S S M K Y I N P P G S F C Q D E L V V E K D E E N G V L L L E L N P P N P W D S E P R S P E D L A F G E V Q I T Y L T H A C M D L K L G D K R M V F D P W L I G P A F A R G W W L L H E P P S D W L E R L S R A D L I Y I S H M H S D H L S Y P T L K K L A E R R P D V P I Y V G N T E R P V F W N L N Q S G V Q L T N I N V V P F G I W Q Q V D K N L R F M I L M D G V H P E M D T C I I V E Y K G H K I L N T V D C T R P N G G R L P M K V A L M M S D F A G G A S G F P M T F S G G K F T E E W K A Q F I K T E R K K L L N Y K A R L V K D L Q P R I Y C P F P G Y F V E S H P A D K Y G W I F Y I T C L R I S

TABLE 7 Variant-3 cDNA CCCGACGTCCTGGCAGCGCCCAGGCACTG Exons 1, Seq ID No 7 TTATTGGTGCCTCCTGTGTCCACGCGCTT 4-11, 11a CCCGGCCAGGCAGCCCTGGCGGATCCTAT TTTCTGTTCCCCCGATTCTGGTACCTCTC CCTCCCGCCCTCGGTGCGCAGCCGTCCTC CTGCAGTGCCTGCTCCTCCAGGGGCGAAA CCGATCAGGGATCAGGCCACCCGCCTCCT GAACATCCCTCCTTAGTTCCCACAGTCTA ATGCCTTGTGGAAGCAAATGAGCCACAGA AGCTGAAGGAAAAACCACCATTCTTTCTT AATACCTGGAGAGAGGCAACGACAGACTA TGAGCAGCATCGAACAAACGACGGAGATC CTGTTGTGCCTCTCACCTGCCGAAGCTGC CAATCTCAAGGAAGGAATCAATTTTGTTC GAAATAAGAGCACTGGCAAGGATTACATC TTATTTAAGAATAAGAGCCGCCTGAAGGC ATGTAAGAACATGTGCAAGCACCAAGGAG GCCTCTTCATTAAAGACATTGAGGATCTA AATGGAAGGTCTGTTAAATGCACAAAACA CAACTGGAAGTTAGATGTAAGCAGCATGA AGTATATCAATCCTCCTGGAAGCTTCTGT CAAGACGAACTGGTTGTAGAAAAGGATGA AGAAAATGGAGTTTTGCTTCTAGAACTAA ATCCTCCTAACCCGTGGGATTCAGAACCC AGATCTCCTGAAGATTTGGCATTTGGGGA AGTGCAGATCACGTACCTTACTCACGCCT GCATGGACCTCAAGCTGGGGGACAAGAGA ATGGTGTTCGACCCTTGGTTAATCGGTCC TGCTTTTGCGCGAGGATGGTGGTTACTAC ACGAGCCTCCATCTGATTGGCTGGAGAGG CTGAGCCGCGCAGACTTAATTTACATCAG TCACATGCACTCAGACCACCTGAGTTACC CAACACTGAAGAAGCTTGCTGAGAGAAGA CCAGATGTTCCCATTTATGTTGGCAACAC GGAAAGACCTGTATTTTGGAATCTGAATC AGAGTGGCGTCCAGTTGACTAATATCAAT GTAGTGCCATTTGGAATATGGCAGCAGGT AGACAAAAATCTTCGATTCATGATCTTGA TGGATGGCGTTCATCCTGAGATGGACACT TGCATTATTGTGGAATACAAAGGTCATAA AATACTCAATACAGTGGATTGCACCAGAC CCAATGGAGGAAGGCTGCCTATGAAGGTT GCATTAATGATGAGTGATTTTGCTGGAGG AGCTTCAGGCTTTCCAATGACTTTCAGTG GTGGAAAATTTACTGGTAATTCTTTATAT CAAAATGATGCCAAGGAGTTGGCATGGCA CTTTGCTAAATGCTGTGTGAATCAATACA AAGATAATTAGGACATGGTTCTTCCTCAC AAGAGGTGTGCAATCTTATTGGGAAATCA TACTTGCAAGTCACAAATATAGACTAAAG TTTCCAGCTGAGAATATGCTGATGGAGCA TGAAACACTAAGGAGACAGGGAGAATCTC AGGAAAAATCAAGAATAATTTGGATCAAA TGGATTCCTGACATAGAACATAGAGCTGA TCAGAAAGAGTCTGACATTGGTAATCCAG GCTTAAGTGCTCTTTGTATGTGGTTCAGA ACAGAGTGTGGGCAGCCTGAGGGGGATAC ATACCCTTGACCTCGTGGAAAGCTCATAC GGGGGAGGGATGAGGCTAAGGAAGCCCCT CTAAAGTGTGGGATTACGAGAGGTTGGGG GGGTGGTAGGGAAAATAGTGGTCAAAGAG TATAAACTTCCAGTTACAAGATGAATAAA TTCTAGGGGTATAATAACAGCATGGCACT ATAGATAGCATATTGTACTATATACTGGA AGTGCTGAGAGTAGATCTTACATGTTCTA ACCACACACACACACACACACACACACAC ACCACACACACACACCACACACACACACG TGCACACAAACAGAAATGGTAATTATGTG AGGTGATGGCGGTGTTAACTAACTTTATT GTGGTCATCATTTAGCCATACATGCATGT CATGAAATCACCATGTTGTACACCTTAAA GTTATGTAATACTAGATGTCAGTTATATC TCAAAGCTAGAAAAAATGTGGGGACCAAG GCAGAAGCTCTTCTGCTCTGTGTCTAAGG GTGGTTCTGGGGCTGGGATGGGGAGGATG GTTAAGTGGTATATTTTTTTCATACCTTT GCTCAGTACTATCATTGTAAGTGTTCAAT ATATGTCTGCTTAATAAATTAATGTTTTT AGTAAAAAAAAAAAAAAAAAAAAAAAAAA AA

TABLE 8 Variant-3 Amino Acid Sequence M S S I E Q T T E I L L C L S P A E A A Seq ID No 8 N L K E G I N F V R N K S T G K D Y I L F K N K S R L K A C K N M C K H Q G G L F I K D I E D L N G R S V K C T K H N W K L D V S S M K Y I N P P G S F C Q D E L V V E K D E E N G V L L L E L N P P N P W D S E P R S P E D L A F G E V Q I T Y L T H A C M D L K L G D K R M V F D P W L I G P A F A R G W W L L H E P P S D W L E R L S R A D L I Y I S H M H S D H L S Y P T L K K L A E R R P D V P I Y V G N T E R P V F W N L N Q S G V Q L T N I N V V P F G I W Q Q V D K N L R F M I L M D G V H P E M D T C I I V E Y K G H K I L N T V D C T R P N G G R L P M K V A L M M S D F A G G A S G F P M T F S G G K F T G N S L Y Q N D A K E L A W H F A K C C V N Q Y K D N

In other aspects of the present invention, nucleic acid constructs are provided that contain cDNA or variants thereof encoding CMP-Neu5Ac hydroxylase. These cDNA sequences can be SEQ ID NO 1, 3, 5 or 7, or derived from SEQ ID Nos. 2, 4, 6, or 8 or any fragment thereof. Constructs can contain one, or more than one, internal ribosome entry site (IRES). The construct can also contain a promoter operably linked to the nucleic acid sequence encoding CMP-Neu5Ac hydroxylase, or, alternatively, the construct can be promoterless. In another embodiment, nucleic acid constructs are provided that contain nucleic acid sequences that permit random or targeted insertion into a host genome. In addition to the nucleic acid sequences the expression vector can contain selectable marker sequences, such as, for example, enhanced Green Fluorescent Protein (eGFP) gene sequences, initiation and/or enhancer sequences, poly A-tail sequences, and/or nucleic acid sequences that provide for the expression of the construct in prokaryotic and/or eukaryotic host cells. Suitable vectors and selectable markers are described below. The expression constructs can further contain sites for transcription initiation, termination, and/or ribosome binding sites. The constructs can be expressed in any prokaryotic or eukaryotic cell, including, but not limited to yeast cells, bacterial cells, such as E. Coli, mammalian cells, such as CHO cells, and/or plant cells.

Promoters for use in such constructs, include, but are not limited to, the phage lambda PL promoter, E. coli lac, E. coli trp, E. coli phoA, E. coli tac promoters, SV40 early, SV40 late, retroviral LTRs, PGKI, GALI, GALIO genes, CYCI, PH05, TRPI, ADHI, ADH2, forglymaldehyde phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, triose phosphate isomerase, phosphoglucose isomerase, glucokinase alpha-mating factor pheromone, PRBI, GUT2, GPDI promoter, metallothionein promoter, and/or mammalian viral promoters, such as those derived from adenovirus and vaccinia virus. Other promoters will be known to one skilled in the art.

II. Genomic Sequences of the CMP-Neu5Ac Hydroxylase Gene

Nucleic acid sequences representing the genomic DNA organization of the CMP-Neu5Ac hydroxylase gene (FIG. 1, Table 9) are also provided. Seq ID Nos 10-28 represent Exons 1, 4-11, 11a, 12, 12a, 13-15, 15a, and 16-18, respectively. Exons 11a, 12a, and 15a are cryptic Exons that are retained in certain variant transcripts of CMP-Neu5Ac hydroxylase. SEQ ID Nos 29-45 represent Intronic sequence between Exon 1 and Exon 4 (hereinafter Intron 1a and Intron 1b, respectively), 4-15, 15a, 16, and 17, respectively. Intron 15a is the 3′ downstream portion of Intron 15 that follows the cryptic Exon 15a. Seq ID No. 9 represents the 5′ untranslated region of the porcine CMP-Neu5Ac hydroxylase gene. Nucleic acid sequence representing the genomic DNA sequence of the porcine CMP-Neu5Ac hydroxylase gene (Table 10, SEQ ID No. 46) is also provided. In addition, contiguous genomic sequence representing the 5′ contiguous genomic sequence containing 5′ UTR, Exon 1 and a portion of intronic sequence located between Exon 1 and Exon 4 (Intron 1a) (SEQ ID No. 47, Table 11) is provided. Contiguous genomic sequence containing an intronic sequence located between Exon 1 and Exon 4 (Intron 1b) through Exon 18 (SEQ ID No. 48, Table 12) is also provided. Nucleotide and amino acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to SEQ ID Nos 9-45, 46, 47, 48, 49 and 50 are provided. In addition, nucleotide and peptide sequences that contain at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90. 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 700, 750, 800, 850, 900, 1000, 5000 or 10,000 contiguous nucleotide or amino acid sequences of SEQ ID Nos 9-45, 46, 47, 48, 49 and 50 are also provided, as well as any nucleotide sequence 80, 85, 90, 95, 98 or 99% homologous thereto. Further provided are fragments, derivatives and analogs of SEQ ID Nos 9-45, 46, 47, 48, 49, and 50. Fragments of Seq ID Nos. 9-45, 46, 47, 48, 49, and 50 can include any contiguous nucleic acid or peptide sequence or at least about 10 bp, 15 bp, 17 bp, 20 bp, 50 bp, 100 bp, 500 bp, 1 kbp, 5 kbp or 10 kbp.

In addition, regulatory regions in the form of putative transcription factor binding sites of the genomic sequence have been identified (see FIG. 4). These binding sites are located in the 5′UTR and Exon 1 of the porcine CMP-Neu5Ac hydroxylase genome, and include binding sites for transcription factors such as, for example, ETSF, MZF1, SF1, CMYB, MEF2, TATA, MEF2, NMP4, CAAT, AP1, BRN2, SATB1, ATF, GAT1, USF, WHN, NMP4, ZF5, NFKB, ZBP89, MOK2, ZF5, NFY, and MYCMAX.

TABLE 9 Genomic Organizational Sequences ctgccagcctaagccacagccacagc 5′UTR Seq ID No 9 aacgctgggtctgagccatgtctgca gcctatgccagagctccccgcagcgc cggatgcttaacccactgagcaaggc cagggattgaaccctcgtcctcatgg atagcagttgagttgtttccacggaa ctcttaggggaactcctgattatttt ttatttaaatttatatttctctgact ttttcgtgtgctcatcagccactgac tgtgtatctccattagtcatggtttg ttaactctgtcattcaaaccctcttc atccttgctacgcagataacatcatt ataataaaatcgtgcctgaagaccag tgacgcccccaagctaagttactgct tcccctggggggaaaaagaagcaccg cgcgggcgctgacacgaagtccgggc agaggaagacggggcagaggaagacg ggggagcagtgggagcagcgggcagg gcgcgggaagcactggggatgttccg cgttggcaggagggtgttgggcgagc tcccggtgatgcaggggggaggagcc ttttccgaagtagcgggacaagagcc acgggaaggaactgttctgagttccc agt CCCGACGTCCTGGCAGCGCCCAGGCA Exon 1 Seq ID No 10 CTGTTATTGGTGCCTCCTGTGTCCAC GCGCTTCCCGGCCAGGCAGCCCTGGC GGATCCTATTTTCTGTTCCCCCGATT CTGGTACCTCTCCCTCCCGCCCTCGG TGCGCAGCCGTCCTCCTGCAGTGCCT GCTCCTCCAGGGGCGAAACCGATCAG GGATCAGGCCACCCGCCTCCT gtgagaaggcttcgccgctgctgccg Intron 1a Seq ID No 29 ctggcgccggcagcgccctccacgca cttcgtagtgggcgcgcgccctcctg cattgtttctaaaagattttttttta tccgcttatgctatcagttactgagg aagtatttacaaatctactattattt tgaatttgcctttttctccttatagt ttatcagtatctcttgagactgttat tggtgcctgcaaatttaaaatgattg gggttttatgaggaagtgaacctttt atctttatgaaacgcctaactgaggc aatgttaattgcttaaaatactttct tattatcagtgtggccatgccagtgt cctcttggttagaatttgcctgat ctgccaaagctgggagatgggggaaa Intron 1b Seq ID No 30 gtagagtgggttattgaaactgaata tagagttcagcatctaaaagcgaggt agtagaggaggaagctgtgtcaacgg aaatactgagctgggttcacatcctc tttctccacacag TCTAATGCCTTGTGGAAGCAAATGAG Exon 4 Seq ID No 11 CCACAGAAGCTGAAGGAAAAACCACC ATTCTTTCTTAATACCTGGAGAGAGG CAACGACAGACTATGAGCAG gcaagtgagagggggctttagctgtc Intron 4 Seq ID No 31 agggaaggcggagataaacccttgat gggtaggatggccattgaaaggaggg gagaaatttgccccagcaggtagcca ccaagcttggggacttggagggaggg ctttcaaacgtattttcataaaaaag acctgtggagctgtcaatgctcaggg attctctcttaaaatctaacagtatt aatctgctaaaacatttgccttttca tag CATCGAACAAACGACGGAGATCCTGT Exon 5 Seq ID No 12 TGTGCCTCTCACCTGCCGAAGCTGCC AATCTCAAGGAAGGAATCAATTTTGT TCGAAATAAGAGCACTGGCAAGGATT ACATCTTATTTAAGAATAAGAGCCGC CTGAAGGCATGTAAGAACATGTGCAA GCACCAAGGAGGCCTCTTCATTAAAG ACATTGAGGATCTAAATGGAAG gtactgagaatcctttgctttctccc Intron 5 Seq ID No 32 tggcgatcctttctcccaattaggtt tggcaggaaatgtgctcattgagaaa ttttaaatgatccaatcaacatgcta tttcccccagcacatgcctaactttt tcttaagctcctttacggcagctctc tgattttgatttatgaccttgactta atttcccatcctctctgaagaactat tgtttaaaatgtattcctagttgata aacagtgaaacttctaaggcacatgt gtgtgtgtgtgtgtgtgtgtgtgtgt ttaccagcttttatattcaaagactc aagcctcttttggatttcctttcctg ctctctcagaagtgtgtgtgtgaggt gagtgcttgtccaaacactgccctag aacagagagactttccctgatgaaaa cccgaaaaatggcagagctctagctg cacctggcctcaacagcggctcttct gatcatttcttggaagaacgagtgct ggtaccccttttccccagccccttga ttaaacctgcatatcgcttgcctccc catctcaggagcaattctaggaggga gggtgggctttcttttcaggattgac aaagctacccagcttgcaaaccaggg ggatctggggggggggtttgcacctg atgctcccccactgataatgaatgag ggattgaccccatcttttcaagcttt gcttcagcctaacttgactctcgtag tgtttcagccgtttccatattaggct tgtcttccaccgtgtcgtgtcgtcaa tcttatttctcaggtcatctgtgggc agtttagtgcgaatggactcagaggt aactggtagctgtccaagagctccct gctctaactgtatagaagatcaccac ccaagtctggaatcttcttacactgg cccacagacttgcatcactgcatact tagcttcagggcccagctcccaggtt aagtgctgtcatacctgtagcttgct tggctctgcagatagggttgctagat taggcaaatagagggtgcccagtcaa atttgcatttcagataaacaacgaat atatttttagttagatatgtttcagg cactgcatgggacatacttttggtag gcagcctactctggaagaacctcttg gttgtttgctgacagactgcttttga gtcccttgcatcttctgggtggtttc aagttagggagacctcagccataggt tgttctgtcaccaagaagcttctgca agcacgtgcaggccttgaggtcttcc gacttgtggcccggggactctgcttt ttctctgtccttttttctccttagtg ggccatgtcctgtggtgttgtcttag ccagttgtttaagggagtgttgcagc tttatgattaagagcatggtctttcc ttgcaaactgcttggtttagaagcct ggctccaccacttagcggctctgtga cctcggacacatttcttagcctttct gggcctcgctcttcttcctcataaag tgaaaatgaaagtagacaaagccttc tctgtctggctactgagaggatggag tgatttcatacacataaagcacttaa aataatgtctggcatatgatacatgc tcaataaatgtcacttacatttgcta ttattattactctgccatgatcttgt gtagcttaagaacagaggtctttaca ggaattcaggctgttcttgaatctgg cttgctcagcttaatatggtaattgc tttgccacagactggtcttcctctcc ttcacccaaagccttagggggtgaac gatcccagtttcaacctattctgttg gcaggctaacatggagatggcaccat cttagctctgctgcaggtggggagcc agattcacccagctttgctcccagat acagctccccaagcatttatatgctg aaactccatcccaagagcagtctaca tggtacactcccccatccatctctcc aaatttggctgcttctacttaggctc tctgtgcagcaattcacctgaaatat ctcttccacgatacagtcaagggcag tgacctacctgttccaccttcccttc ctcagccatttttcttctttgtacat aatcaagatcaggaactctcataagc tgtggtcctcattttgtcaatctaat ttcacagcctcttggcacatgaagct gtcctctctctcctttctgcctactg cccatgagcagttgtgacactgccac atttctcctttaacgacccagcctgc tgaatagctgcatttggaatgttttc aatttttgttaatttatttatttcat cttttttttttttttttttttttttt ttttttagggccgcacccatgggata tggaggttcccaggctagggatccaa tgggagctgtagctgctggcctacac cacagccacagcaatgcacaattcga gccaatctttgacctacaccagagct cacggcaacactggattcttaaccca ctgattgaggccagggatcaaactct cgtcctcatagatacgagtcagattc gttaacctctgagccatgatagttgt tagttactcattgatgagaaaggaag tgtcacaaaatatcctccataagtcg aagtttgaatatgttttctgccttgt tactagaaaagagcattaaaaattct tgattggaatgaagcttggaaaaaat cagcatagtttactgatatataagtg aaaatagaccttgttagtttaaacca tctgatatttctggtggaagacatat ttgtctgtaaaaaaaaaaaatcttga acctgtttaaaaaaaaaacttgactg gaaacactaccaaaatatgggagttc ctactgggacacagcagaaatgaatc taactagtatccatgaggacacaggt ttgatgcctggcctcgctaagtgggt taaggatatggtgttgctgcagctcc aattcaacccctatcctgggaacccc catatgccaccctaaaaagcaaaaag aaaggtgctgccctaaaaagcaaaaa gaaagaaagaaagacagccagacaga ctaccaaatatggagaggaaatggaa cttttaggccctatctccaactatca catccctatcaccgtctggtaagaaa tggaaaaaatattactaagcctcctt tgttgctacaattaatctgattctca ttctgaagcagtgttgccagagttaa caaataaaaatgcaaagctgggtagt taaatttgaattacagataaacaaat tttcagtatatgttcaatatcgtgta agacgttttaaaataattttttattt atctgaaatttatatttttcctgtat tttatctggcaaccatgatcagaaat ctttaaacaatcaggaagtctttttt cttagacaaatgaaaatttgagttga tcttaggtttagtacactatactagg ggccaagggttatagtgtgactatta aatcacagataatctttattactaca ttatttccttatactggccccacttg gatcttacccagcttagcttttgtat gagagtcatccttaaagatgacttta ttctttaaaaaaaaaaacaaatttta agggctgcacccatagcatatagaag ttcctaggctagcggtcaaattagag ctgcagctgccagcctatgccacagc cacagcaatgccagatctgagctgca tctgtgacctacactgcagcttgcag caatgctggatccttaacccattgaa caatgccagggattgaacacacatcc tcatggatactgctcaggttcctaac ctgctgagccacagttggaactccaa agcagactttattctgatggctctgc tgatctctaacacgttattttgtgcc atggtgtttatcttcactttactcaa gtcagggaaacacgaagagtctcata caggataaacccaaggagaaatgtgc aaagtcacatacaaatcaaactgaca aaaatcaaatacaaggaaaaaatatc ttcactttcaaaatcacctactgatg atgagtttatatttccttggatattt gaatattagctatttttttcctttca tgagttttgtgttcaaccaactacag tcgtttactttgatcacagaataatg catttaagccttaaatagattaatat ttattttcaccatttcataaacctaa gtacaatttccatccag GTCTGTTAAATGCACAAAACACAACT Exon 6 Seq ID No 13 GGAAGTTAGATGTAAGCAGCATGAAG TATATCAATCCTCCTGGAAGCTTCTG TCAAGACGAACTGG gtaaataccatcaatactgatcaatg Intron 6 Seq ID No 33 ttttctgctgttactgtcattggggt ccctcttgtcaacttgtttccaatct cattagaagccttggatgcattctga ttttaaactgaggtattttaaaagta accatcactgaaaattctaggcaagt tttctctaaaaaatcccttcattcat tcatttgttcagtaagtatttgatga gaccttaccatgtgtaaacattgcac taggtattaagaaatacaaagatgga taagatagagtcggcgtaaatgagat gatataatgagacgttataatgaaac tcacaattccagttgggaaataaagt ccttcaaattccatgactctttctgg cacacgttagaggctacagcttctgt gtgattctcatgctggctccacttcc actttttccttcttcctactcaagaa agcctatagaaatatgagtaagaagg gcttaatcataggaataaatttgtct ctgttctaagtgattaaaaatgtctt tatcagtataaaaagttacttgggaa gattcttaaaactgcttttacacact gttctagaatgactgttatataaata aaaaagtagatttgatctaacacaat taaatgacctttggaaatattgacta attctcaccttgcccctcaaagggat gcctgaaccatttccttcttttgcca gaaagcccccaccctttgtctgttga cctagcctaggaaatcttcagatcac gttgttagcacgaactggttacatgt gctgtacaaatactatttaattcatc tgattaaaaaaaaagagataagaagc aaaagtttgactatcttaaactgttt gcgtaggtgagaggacaattgaccat ctactttatgagtatgtaacccagaa acttaaagctccttaagggagctaag tcttttggataagacctatagtgaga ccttttagcaaaatggttaagactga atggagctcactagcgtgggttcata tcctgatgctcaaacacgcaattaaa tgactttaggtgggttagtctctgtt ccttagtttcctcaatgggagataat attggtagtagcgattttactgggtt gttgaaagaacatctgttaaatgttc agaacgtgttacgacagagtacagag taatgatttgcttgtatatgtatgac tcaaatagtctgccatatgccttgtg actgggtcctgtggagcaggaaggag ggatttcccacccagcagaaagttgg gtaaactggaaaatagactgaggcca ggaaatgatgcaaagcgttgatgttc actgccacggcaggtgaagggcaggg ccagagttgtcagtagggtcagggga ggactggaaataaccaagacccactg cacttttcagcctttgctccagtaag gtaatgttgtgagagtagaaaatttt gttaacagaacccacttttcagtaca gtgctaccaatactgtagtgatttca taccacatcccaagaaagaaaaagat ggctcaatcccatgtgagctgagatt atttggttttattgttaaataaatag cattgtgtggtcatcattaaaaaagg tagatgttaggaaagtagaaggaaga agactctcacctacattttcatcact gttttggtatctgccagttgtcacct tggtccccttccccgcctctcccctg cctcctcttcctccttctcctttttt tggaatacaattcaggtaccataaaa tttacccttttagagtgtttgactca atggtttttagtattttcacatgttg tgctattactatcactatataattcc aggtcattcacatcaccccccaaaga aaccttctaactattagcagtccatt cccttcttccctcagcccctggcaac cactaatctacttactgtctccatgg atgttcctatattgaatcaagctagc ataaaccccacttgctcatggtcata attcttttttatagtgctaaattaca tttgctaatattcaattaaggatttc tatgtccatattcataaggaatattg gtgtgtagttttctctttgtgtgata tctttgtctggttgggggatcagagt aataattactgctctcatagaatgaa ttgagaagtgttccctccttttctat ttattggaagagtttgtgaagtatat tggtattgattcttctttaaacattt ggtcagattcaccagtgaagccatct gggccatggctaatctttgtgaaaag ttttttgattactaattaaatctctt taatttgttatgggtctgctcctcag acgttctagttcttcttgagtcagtt ttgttcatttgtttcttcctaggact ttctccctttcatttggattatttag attgatagtaatatcccccttttaat tcctggctgtagtaatttgggtcttt tctcttttttcttggtcagtttagct aaaggtttgtaattgtattaatcttt tcaaataactaacttttttgttttgt ttgttttttgttttttgttttttgtt ttttgtttttttttgctttttaaggc tgcacctgaggcatatggaagttctc aggctagaggtctaatcggagctaca gctgctggcctataccacaaccatag caatgccagattcaagctgcatctgc gacctacaccacaactcggccaggga tcacacccgcaacctcatggttccta gtcggatttgttaaccactgtgccac gacgggaactcccgcccatttttttt aacacctcatactttaacataaagat gggcttcacatggactgatagctcaa atgaggaaggtaagactatgaaagta atggaagaaatgtagactatttttgt gacctagagattactgatacttcttg acttttcaaacaatacttcaaaagta cagcccaaagggaaaaaagaaagaaa aaagaaacacacatatacacaaacct agtgaataagatatcatcgatacact acagatttctatgaactggaagaccc catggacaaagttaaagaacatatga tagtttgagtgattattttgcaatat ttacaaccaatgagggaatattatcc agcttataggaggaagtaatgcaaat cgacaagaaaaagataggaaacccaa tataaaaattaagaaaatacaaaaat taagaaaggatatgaactagcatttt acaaaagaaaaatctccaaaagtcaa tcagcacatgaaaatatgctcaaacc taattattagaaaactacagactgaa gcaatgaggtgctttactttacatct ttttgactgataaaaagttagaaaca aaggtgatatcaaatgtcagggataa aaggatatagaaatcgtcatgcctgt ggtgggagtatggccggtgcagtcat gtgggaaggtaatctgacagtggtta ggcagagcaggtttatgaatacactg tggcccatcaatcccacgcctgttta tgtaccaaagaaatcctgttgtggca gaatctatgggtccacccctgggagc atgaattaataaaatgtggcaccagg gtgtgtgaaactccagctagagatga gatgtccacatggcaacatgaatgca tcttagaaacatagatttgagtgaaa aagagtaagaaacagccgggaaaccc aataccatttataaaaattaaagatg cacacatacaatgtagtaaatatttt gcatgaactttcaaatggttgcctac agggggggagagtaaagaagagtaga aaacaaagataaagggagtaagtaag tagctctgcctggactgaatataatg tgtcatgaactgagaaatatggttaa cataatcctcttaacttgaggtccta aatgaatgaatgagtccactattcat ttacccattctttaatgtgtattgca ttataatccatttttttagaaccaac gaattttgttcccataactactaatc agcctgccttttctccctcattccct tatcagctcaggggcattcctagttt ttcaaacgttcctcatttgaaccaaa aatagcatcattgtttaaattatact tgttttcaaatacgatgcttatatat tccaagtgtgtttgcccattttctta ggtggtagaaatttttcattctactt ttctatctactcagattttcccgttg gaattatttccattgctattaaactt agaagtcccccctgtgatatgccatt tttttcatactttttaagcacttggt tgcttttctttgtgtctttaagcacc tagaatacttataaccattgcacagc actgtgtatcaggcagcccttcctct tccactaatttatggtccttctctta gactatattaaactgttatttaatta ggatcctctcttcgtccttatgattt aattattatagttttctaatatgttt ttattataattcctcttcattattcc tccctattaaaaattttaatgaattc catttgtttgttcttctagttaaata ttaagtcataatccaaataacttaga tgtcattagtttatgtggtcaaagta aggataccacatctttatagatgcag gcagttggcagatgtcatgattttct tcagtgcataaatgcaatttatcttt gagcaaggggcataaaaacttttatg gtattggctttgaaataatagttaag aactgcagactcagtttttcctgctt ttcttgaaaaagaacacttctaaaga aggaaaatccttaagcatggatatcg atgtaattttctgaaagtctcctgta attccttgggatttttgttgttgttt gttggtcggtttttttgggtttttgt ttgtttgttttgttttgttttgtttt gcttttagggctgcacctgtggcata tggaagttcccaggctaggggtccaa ctggagctacagctgccagcctactc cacagccacagcaacatgggatccta gctgcatctgtgacctaaccacagct cttggtaatgccagattgttaaccca ctgagcaatgccagagatcgaatctg cctcctcatggacactagtcagatta gtttctgctgagccacaatgggaatt cccaattccttgtatttttgaactgg ttatgtgctagcatataattttgttt cttgaatctttgtgggtttttttttt tttttttttttgtctcttgtcttttt aaggctgcacccacagcatatggagg ttcccaggctagaggtcaaattggag ctacagctgccagcctacacaacaac tgcagcaaagtggggcccaacttata tgacagttcgtggcaatgccggattc ctaacccactgagcagggccagggat cgaacctgagtttccagtcagtttcg ttaaccactgagccatgatagtaact cctgtttgttcagtcttgaacctcct ttttaattctttattccttgagggtg aaataattgccataataatactatca tttattacatgccttctctgtgctag gcatagtgacactttaggatttatta tatcacttaatccctacaacaactct gcaaagtatgtatcataatcctattt gacagatcaggaaattgcagcccagg atgcagataatatgcatccatcacaa gtgactagatatagtccctctgctat tcagcagggtctcattgcctttccat tccaaatgcaatagtttgcatctatt gtatatgtgttttggggtttttttgt ctttttttttttttttgtcttttctg gggcctcacccttggcataggtaggt tcccaggctaggggtcaaattgaagc tgcagctgccagcctacaccacagcc acagcaactcgggatctgagcctcat ctgcaacctacaccaaagctcacggc aacaccggatccttaacccactgagt gaggccagagatcaaaccggcaacct catggttcctagtcggattcattaac cactgagccacgatgggaactcccta aatgcaatagtttgctctattaaccc caaactcccagtccatcccactccct cctcctccctcttggcaaccacaagt ctgttctccatgtccatgattttctt ttctggggaaagtttcatttgtgcca tttttcattttacgggtaatttttac ttcagtttcttccactagcagttgtc ttaaagtgagtataattaatattcat ttggaaaatgtaagcaaaacattttt taaagggccatgcccacagcatatga aagtttctgggccaggggttgaatcc aggctccaagttgcagctgtgcccta cactgcagctgggcaatgctggatcc tttaacccactgtgcccggctaggga tcaaacctgcatttccacagctaccc gagccattgcagttggattcttaacc cactgcactacagtgggaactcccac aaaacattttttaatgtcctttgaat aaagtaggaaagtgctcgtctttgag ggcagggcggcaatgccatttccaca aggtttgctttggcttgggacctcat ctgctgtcatttagtaatgaataaaa ttgctgacagtaataggattaactgt gtgtggagatagccagggttagagat aaaaacactggagaagtcaaataagt tgctcgaggtcctctagctaataagc tattaagtgggagagtgagggctaga aacaggccatctgtctcccaagcaca tgtccattagtggtttgctgatagcc ttccagaacaacagagaggactctca aacatggtcttgcctccctccaattg atcccctccatgtgcctcacagcggg tctttctaaaattaagttctgatttt aattctcccttgctatagcacttagg tatggctttcagccgtgcaataaaaa gcaggcaagagtggctcaatcatata ggaggttgtttttcttagatcccaag caggtaatcctgggcattatggttgt tctgcgtttatcaaggagccaaattc tctatcacctcctgttctatcctcct cagtatctggctctattcttcagcat ctcaagatggcttgtgctcctccaag catggcagtcaaattccacacaagag ggggaaatatgaagggcagacagtgc tggtctcctgagctgtccctctttgt cggggaaataaatgtattccttcaag tcccgtgagacttctgaagtagacgt ctgcttacgtctcacccaccagaact atgtaaactgcacatagtgctaggtc tacatagccactcataactgccaggg ggtgggaaatctttaaataggtgtac caccacacaattaggatgctaatagt aagggagaaggagagaataggttttg cgcaagccaccagcatgcctgccaca attgcttaaaattcttcattgacccc tcattgccacaggatgaaatccaaac gccttcttagttgggaatctgaccta cctgtctctcccacctggttcagaca ccattctccttggtcataaaattcca gtcatttgtgaacatccagctccccc atgcctccatgcctttgcacatgctg ttcttttatcttttatgttgtccttt tatcttttatccaaaagagatatccc atcatcacatctcttttgtcagcccc caaatactttgtctttcaagttcagc tggaggattacctcctatttgaaatc agctttgtctcttacaaccaaacaag gttttccttccgagacactcccacag caccttgaactcatctctatcaatca ttcatttgataatgaagttgttggtg gtatgcctgtgtctctgacacatctg cgatctcatgagttccttaagtggaa tgtgaatagcgggatgaacagtattg gtcttcagccctcatctctgcagatg ttgcttgacccaaatgagcgttgcct tttattttgattttgctttgatttgt ctactccatgtacttgagccatgcat ttctgtcttagcgatgctttttaaaa gtcattttttggttgattatccagat ttgtccacctttgcttctag TTGTAGAAAAGGATGAAGAAAATGGA Exon 7 Seq ID No 14 GTTTTGCTTCTAGAACTAAATCCTCC TAACCCGTGGGATTCAGAACCCAGAT CTCCTGAAGATTTGGCATTTGGGGAA GTGCAG gtaaggaaatgttaaattgcaatatt Intron 7 Seq ID No 34 cttaaaaacacaaataaagctaacat atcaatttatatatatatatatatat atatttttttttttttttacatctta tattaccttgagtattcttggaagtg gctagttaggacatataataaagtta ttctgaagtctttttttttctttttc catggtgagcagtggcttgatgtgga tctcagctcccagacgaggcactgaa cctgagccgcagtggtgaaagcacca agttctagccactagaccaccaggga actccctattctaaattcttgagcac attatttaggaacctcaggaacttgg caggattacaggaaatatatctagat ttaaaaaaaaatcttttaacagaggt cccaaaggagagtcatgcacagctat gggaggaagttcagaaactgcccttg ctaccagatcactgtcagataaaatg gccagctacatgtttctgcacattgc cctaagatctttacaaacttttctgt gcatttttccacttttaaaagaaaat ttcggggttcctgttgttgctcagtg gttaacgaacccaactagtatccatg gggacaggggttcgagccctggcctc actcagtgggttaagaatctggcatt gctgtggctgtggcgtaggctggcgg ctacagctcagattggacccctagcc tgagaacctccatatgccgcaggtat ggccctaaaaaaaaaaaaaaagagag agagagaatttcctccagaaaaaaca ctttggtagtttgggagaagtaaaca accaaaaattaatttttctggagtat tcgggaagcttgtaaaaatgggctct tacttttttgaggagacaaatgggaa cctacccagaagaggcacaatcacct gcatttgatttcttgacctctcccta ccttctttgctggctttccacatttg gatttctgtgaccttatctctgctcc ttggtgttttcatttttcctgtggac gtgccagactatgggaagggagtaag gcgttgatttagaatcctgtagtctc tgcctgtctctagtcattgttttcac ccttctcaaaggaccttgacatcctg agtgagtccgcaagtaatttagggga gaagccttagaagccagtgcagccag gctacatgactgtgtccacccactgg aaccagtcatttttatacctattcac agcccccctaccatttaaatccccag aggtctgccataacatctgtaactcc ctttcctggtaaattgtgttctaaaa gactggtaacaaaagatattctgtgg tacagagcataattaaatacctggga gctgatttgagtggggtaaatcaact ggtttgacccctaaaacccaccatga gcatttctgttctaataaagtaatgc ccgtgctgggaagttctacggaaatg ctcctgctgtgtctttcttgagtcct gtgtcattgaacatgcttaggagcaa aggtcccccatgtggcttgtctgcta accagcccagttccttgttctggctg gtaatgatccgatcatctgaatctca ctgtcttccaacag ATCACGTACCTTACTCACGCCTGCAT Exon 8 Seq ID No 15 GGACCTCAAGCTGGGGGACAAGAGAA TGGTGTTCGACCCTTGGTTAATCGGT CCTGCTTTTGCGCGAGGATGGTGGTT ACTACACGAGCCTCCATCTGATTGGC TGGAGAGGCTGAGCCGCGCAGACTTA ATTTACATCAGTCACATGCACTCAGA CCACCTGAG gtaaggaagggtgagccctcaactcc Intron 8 Seq ID No 35 gaagaaaatgctgcaataaaagcact gttggttttcagctttttttgtaatc actgctcattctgaggtagattcgct tgggctgataaaaagagaactaattc agataaatgcttgcatttgcatagcc tctttttttaaaaacttttttttttt ttttttttttttggcttttcagggct gaacctgtggcatatggaggttccca ggctaggggtcgaatcagagctgtag ccccgggcctatgccactgccatagc aacatgcatagcctcctttttaaagt gccttcctgttttataccattgggat gtgagaagagctattgtggaaangag catggggtnataaccctggacctctc acgtcctaccctcaggntagtgggaa aactctgagtttaaggacatcaaagt gactcctttttagttacattatggng gaatcagcncatatttttacaagggg cggagngtaanctgttggagtttaca agacatatggtggcattgcaactact taaccctactattatagcacaaaagc agccatagtcggtcctgaaggagcct gatgccttcagctttataggcaatga cgtgtgaatatcacaaacagtttcct gtgtcaccaaacatgattgccttttg atttccctttcaaccctttaaaaaaa ggtaaaagcccttcttagcattcagc agcaggtcgctgtgttttgccaactc ctgatctgtagcatttcgacaacact gagctctcaacttttgaaccctgagt ccaccacatccttcagtgaaaccaga gccatgtgatactaaggatagaaacg gaaacttcctgaatccaggcgatcaa ataggagggagaaagaggaactttca ttgacaaaaccacaaatattgtgaat ggactgttacaaatattgtgaatgct cctattcccaaccccctggcttcatt acagggtcctatgtgttcatccttat tgagaaatttgtattgctactgccag gttgccaatacccagcggtgcccatg gtgttctaaaatgaagcaatttcaac tttatttttttttcctgtgactttac atgacaagttcacatgaaggatatac tttgatagtaatgtccatggttaggg aatatacattgtttgctggttgactg gcccctggatttttctattgaaagtc catgagatctcgaaggcacaggtgtg ttctctcgctttttaaggaaagggtt taaaaacttaagtaattaacagcttt agtaacaaattacctataacacactt aaaaaccgaataccacccactggagt attgtgctacgattaaaaatctactt gtctactacatgatatctttgtccca cagaaggttctggaaccaaacttgta atttcaggattatgagagccctgagt tcacgcattgtgtaataactatgttg tgtggtagtcaatttgtacagcttgc ttagagagaacaatgtcaagttaagg aggcgattgctttatagtgcctgtca caagatgccattgccattgtcctagc aagagatattctatgggagtatacta cattttagtgaggataagaacttttt atggcatttagtccggtcatttccca accactgtcctgaaaaccaatttcat tttgatttcaggggcttgtgtgggca aagttgccaggcattaaaaagccact tctcaactgtagtatcacaatgcttt agttgggtagtgtattgcagatagct tatggctgaaaagttaccaagccttg cagttttcactcctttgagtttattt ccttgacagaattgaccctgagtttt ttgactcttacctgctcaactaataa acaccagagtcatttatctccattgc tcttgtctgacctttatttaccgaat aatgccttatgggttcacaaaaacaa ggggggagggggccagcatgccttag aaactgtctttagtcaagaaatgnga ttttattatgtaaatatatgagtatt ataatagatagtgttattaatagaca ccagcaagaattgtcaataatttaaa aatcacaaattaaaatacatccatgt tagnatcatttatcctaactcccaaa gccctttaaagtggaagatttagatg ttaacccagagattaaagacatgttc aaagaatccttgatttttttttgaat cccttgtttttagagaagaaaaccta atgattttccccctctggattctaca tattaaatatagttttggaacttgaa tattagtatggttaataagtgctgat atgctgattttgtttatatttttctt atgagtaaatatcctatatcaccaga cattatagtctatgtacaaatatgat tcttaaacctgatagcacattcatta gagttggaattgcctttttttttttt ttttttacagttgcacctgcaacata tgaaagttcccaggctaggggttgaa tccaagctgcagctgccaccctacat tacagccgtagtaacagcagatccga gctgcatctgcaacctatgctgcagc tcagggcaatgccagatccactgagt gaagccagggatggaacttgcatcct catagagacaacgtcgtgtccttaac ccactgagccagaacaggaactccag aatttcctttcaatagaagaagcacc aagtttaggatcagaaagcctgaatt tgaataccaatttactatttgttagt catatatttctgagtgtgtttcctca tttattaaaagcagactaaaagatga gagggtcttttgttgagaatcaaata caataacatgtgaaagtgtgtaacac tatgattgaaatatacctacacagcc atttatttgtttattgttcatgtttt gccacccacacagtagtatataatcc ttttatgtaataaatgctaataatga aagttggcaacttatgtaagtactca aaatgctggaggtcatgggatactga ctgggatactacagaggtaatgtcat ttcctctgcgctaaacttattgtctt gtagttagggactgactctctttagg acaaggagttcattctgtataccatg tgtggctatcacccttcgaagttgaa aaactgccccagggtgggcacccatc cgttctcttagatatatggccgagac ctttctctcactgggagggaaccaca ctgaggaatgagaaaaaaaaaaggaa aatcaagatgaaaccagaaacctctt tggcataacttctccactctgtactt tttgttagaactacccttgcacaaag cagcatcagtgtggaagacagaattt gcacacctggtttgatatacatgccg tggtatatgggatgttctaacaataa agaggactctcccaggaaatctcctc actgttatagtcagccttgaggaaag agctcttcttttggactctggggaga gtctagtttttcagttccttgcttct cggtcaacgtgttggtgtaaggatca cactctctcttatactagataattct attttttcacctttcaacctgtctat ccttctgaccctag TTACCCAACACTGAAGAAGCTTGCTG Exon 9 Seq ID No 16 AGAGAAGACCAGATGTTCCCATTTAT GTTGGCAACACGGAAAGACCTGTATT TTGGAATCTGAATCAGAGTGGCGTCC AGTTGACTAATATCAATGTAGTGCCA TTTGGAATATGGCAGCAG gtctgtgttctttccacatgtttggg Intron 9 Seq ID No 36 ttatcctttctgggataaatttgagg cgagatagaaactttaagactaaaga aacaatggcctactttttttgtacat ggtcctgtgtaaatctctatttgagc tgaaataagatggtcttcctctccaa ttatccatggtatgactctgatggat aacaaatccagttctgaaaaaagggg atttctttccagaagagaggacagtt tcttcaaatattgaattaaaagcaaa atagatgtaaaccgttgttggtttta ttgttgaattccag GTAGACAAAAATCTTCGATTCATGAT Exon 10 Seq ID No 17 CTTGATGGATGGCGTTCATCCTGAGA TGGACACTTGCATTATTGTGGAATAC AAAG gtattttcttgccctcatcagcatga Intron 10 Seq ID No 37 aattgctcttggtagaaaggataata atagttatccaaaacatcatcctatg ttcatctgtttcttccctcttcattt tccatagagtacagtatattctatct ctgtcttaggaaaatggactgtcatt catataatcttacagagaatcaatta gtaatgtactctatgccgtgacaggt gcgaaggttttttttgaaggcaacag ataaaaatatcctatatttcacctat tgtaatttccttaaaactgacattat tgaataaatgttttactttcatcttg aatattattatgttatggaatcatac actttaccccaataatcatcgaaaag aatttccaaaaggttgagagagttgt gttgatctgattactttcctctgcat cctttgagcttaacctttgaatatag tttgctaaggaaagtagtctgtttat gatcctggagtggaatcaggctaagt gtcctcattcagaacccactgaatca gacagaatgaatttatttccttgaaa gttcaaaatgtgtcactcagagtata aattttcaaatcttactctctctttt ccttggatgtgagcaattcttcgata attgaatgaggcagattatatagact tacatggaagactgttggcctgagaa ttcaaactatggtgttcaagacttca cngngagtccgatgccatttgtttcc cacag GTCATAAAATACTCAATACAGTGGAT Exon 11 Seq ID No 18 TGCACCAGACCCAATGGAGGAAGGCT GCCTATGAAGGTTGCATTAATGATGA GTGATTTTGCTGGAGGAGCTTCAGGC TTTCCAATGACTTTCAGTGGTGGAAA ATTTACTG GTAATTCTTTATATCAAAATGATGCC Exon 11a Seq ID No 19 AAGGAGTTGGCATGGCACTTTGCTAA ATGCTGTGTGAATCAATACAAAGATA ATTAGGACATGGTTCTTCCTCACAAG AGGTGTGCAATCTTATTGGGAAATCA TACTTGCAAGTCACAAATATAGACTA AAGTTTCCAGCTGAGAATATGCTGAT GGAGCATGAAACACTAAGGAGACAGG GAGAATCTCAGGAAAAATCAAGAATA ATTTGGATCAAATGGATTCCTGACAT AGAACATAGAGCTGATCAGAAAGAGT CTGACATTGGTAATCCAGGCTTAAGT GCTCTTTGTATGTGGTTCAGAACAGA GTGTGGGCAGCCTGAGGGGGATACAT ACCCTTGACCTCGTGGAAAGCTCATA CGGGGGAGGGATGAGGCTAAGGAAGC CCCTCTAAAGTGTGGGATTACGAGAG GTTGGGGGGGTGGTAGGGAAAATAGT GGTCAAAGAGTATAAACTTCCAGTTA CAAGATGAATAAATTCTAGGGGTATA ATAACAGCATGGCACTATAGATAGCA TATTGTACTATATACTGGAAGTGCTG AGAGTAGATCTTACATGTTCTAACCA CACACACACACACACACACACACACA CCACACACACACACCACACACACACA CGTGCACACAAACAGAAATGGTAATT ATGTGAGGTGATGGCGGTGTTAACTA ACTTTATTGTGGTCATCATTTAGCCA TACATGCATGTCATGAAATCACCATG TTGTACACCTTAAAGTTATGTAATAC TAGATGTCAGTTATATCTCAAAGCTA GAAAAAATGTGGGGACCAAGGCAGAA GCTCTTCTGCTCTGTGTCTAAGGGTG GTTCTGGGGCTGGGATGGGGAGGATG GTTAAGTGGTATATTTTTTTCATACC TTTGCTCAGTACTATCATTGTAAGTG TTCAATATATGTCTGCTTAATAAATT AATGTTTTTAGTAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAA gtaattctttatatcaaaatgatgcc Intron 11 Seq ID No 38 aaggagttggcatggcactttgctaa atgctgtgtgaatcaatacaaagata attaggacatggttcttcctcacaag aggtgtgcaatcttattgggaaatca tacttgcaagtcacaaatatagacta aagtttccagctgagaatatgctgat ggagcatgaaacactaaggagacagg gagaatctcaggaaaaatcaagaata atttggatcaaatggattcctgacat agaacatagagctgatcagaaagagt ctgacattggtaatccaggcttaagt gctctttgtatgtggttcagaacaga gtgtgggcagcctgagggggatacat acccttgacctcgtggaaagctcata cgggggagggatgaggctaaggaagc ccctctaaagtgtgggattacgagag gttgggggggtggtagggaaaatagt ggtcaaagagtataaacttccagtta caagatgaataaattctaggggtata ataacagcatggcactatagatagca tattgtactatatactggaagtgctg agagtagatcttacatgttctaacca cacacacacacacacacacacacaca ccacacacacacaccacacacacaca cgtgcacacaaacagaaatggtaatt atgtgaggtgatggcggtgttaacta actttattgtggtcatcatttagcca tacatgcatgtcatgaaatcaccatg ttgtacaccttaaagttatgtaatac tagatgtcagttatatctcaaagcta gaaaaaatgtggggaccaaggcagaa gctcttctgctctgtgtctaagggtg gttctggggctgggatggggaggatg gttaagtggtatatttttttcatacc tttgctcagtactatcattgtaagtg ttcaatatatgtctgcttaataaatt aatgtttttagtaagtaatctctgtt tagtaatgtgtcagaaatgccctact tgcaataggaagaaaacctgtccagt cccttccttttttctgtaagtctgat ttcattgcctcccagaatgcatcacc atgtgagagatagagggaaggtgctg tccttatggggttaacagtgtgacta gggaggcaaaatatacctactaaagg gtggtagcataattcagttcttatgt gagtatgtgtatgtgtgtgagtatgt gcacatgcacatacattttaaaaggt ctgtaatatactaacatgttcatagt ggttacacctagcttataggtaacat tttttcccctgtatccttgtttgtgt ttatcaaattttcataacagtaatgg tagaaggagtacctgacatggtacca tacatgctnggncctgcctaatttct cnatttcctttattgcccataccccc attgcttgacaagcataagtccatac tggcttgttttcgttcctcagactca gtacaccatgtagctccatgccctgg gtctttgtatgtgctatttctactgc ttagagtgctattgcccctgaccacc acgtggtcagcaacttctcttctgcg tctgtgtctatggtctatgattccag atgtcatcttcactaactacccttct aatatgcccttccatcccacccgtcc tcatccttaccccagccactctctat ttggtggctctgttttattttcttcc tagctcatcactctttgaaatgaact tatttacttattcaatatttgcttct ttcactagaatgaatgctccatgaga gcagggacctgctttatcttgctcgc cactgtattcacagtgcctagaacta cgtctggcacatagtaggtgctcaat aaatatcgatcaaatgaaagaatgag caaacgaacaaatgaacaacacgtga ggtaggcatcatgattccatcaacag aggagaaaaccagacttaaagnaatg aagtggnggagctgcatttgatcttg actgactccaacatccatgctcttga ccactgtgcatctccagagtgtaatg aacatactttacttttatattccacc aaaataacaaagccatgcccatgtta gtagagagttaatcgacagtgccctt aaaatatgcatgcacccagggtacaa ctatgcatgctgccctgtgttttcag ttggatccaaatgaattgccgtaaac aaagaggggattcaatgtctttgact agtttgggatattttcctagtaacca actttgcaaaataaagccactaatga caaggagctttgttctacttctgcat cactcaactgtcaatttttatctctt gcaagacttctaatctactagaactt ttgtttttctgtgatttctgaacaga gaagactaatccaaaccctgtcattc cag AGGAATGGAAAGCCCAATTCATTAAA Exon 12 Seq ID No 20 ACAGAAAGGAAGAAACTCCTGAACTA CAAGGCTCGGCTGGTGAAGGACCTAC AACCCAGAATTTACTGCCCCTTTCCT GGGTATTTCGTGGAATCCCACCCAGC AGACAA GTATGGCTGGATATTTTATATAACGT Exon 12a Seq ID No. 21 GTTTACGCATAAGTTAATATATGCTG AATGAGTGATTTAGCTGTGAAACAAC ATGAAATGAGAAAGAATGATTAGTAG GGGTCTGGAGCTTATTTTAACAAGCA GCCTGAAAACAGAGAGTATGAATAAA AAAAATTAAATAC gtatggctggatattttatataacgt Intron 12 Seq ID No 39 gtttacgcataagttaatatatgctg aatgagtgatttagctgtgaaacaac atgaaatgagaaagaatgattagtag gggtctggagcttattttaacaagca gcctgaaaacagagagtatgaataaa aaaaattaaatacaagagtgtgctat taccaattatgtataatagtcttgta catctaacttcaattccaatcactat atgcttatactaaaaaacgaagtata gagtcaaccttctttgactaacagct cttccctagtcagggacattagctca agtatagtctttatttttcctggggt aagaaaagaaggattgggaagtagga atgcaaagaaataaaaaataattctg tcattgttcaaataagaatgtcatct gaaaataaactgccttacatgggaat gctcttatttgtcag GTATATTAAGGAAACAAACATCAAAA Exon 13 Seq ID No 22 ATGACCCAAATGAACTCAACAATCTT ATCAAGAAGAATTCTGAGGTGGTAAC CTGGACCCCAAGACCTGGAGCCACTC TTGATCTGGGTAGGATGCTAAAGGAC CCAACAGACAG gtttgacttgaatatttacagggaac Intron 13 Seq ID No 40 aaaaatgatttctgaattttttcatg tttatgagaaaataaagggcatacct atggcctcttggcaggtccctgtttg taggaatattaagtttttcttgacta gcatcctgagcttgtcatgcattaag atctacacaccaccctttaaagtggg agtcttactgtataaaataaactatt aaataagtatctttcaactctggggt ggggggggagactgagttttttcaca gtcctatataataattttcttatcct ataaaataattaggagttcccgtagt ggctcagcaatagcaaacccgactag tatcgatgaggatgcgggttcgattc ctggcccccctcagtgggttaaggat ctggcattgccgtgagctgtggtgta ggtggcagacacggctcagatcccac gttactgtggctgtggcataggccag cagctccagctctgattagaccctta gcctgggaacttccatatgctgtggg tgtggccttgaaaaaaaataaataaa taagataattactcaaatgttttcct tgtctcagaaccttacttcaggataa agagtgagaaagttttttttatgaag ggccattattacagctcaaaaataag ttgtcttcagcaagtagaaagcaata agcctgagagttagtgttcctatcag tgtaaatattacctcctcgccaatcc ccagacagtccatttgaacaattaac ggtgccctgggagtacagttcagaaa cattaatgtggatgttccagacctgt atttttataagtacttgtcttgagcc ggatggaaccatcattcctcaccatt atttagaagtggactgtgactctgtt ggagatcagggcacacggttaccaaa agcacacccttctcctggccttacct ttgcaaagctggggtctgggacacag tcagctgattatacccttttactaac ttcccacagctcaaatctggtcaatt ctccttcacaaatctcttaaaaatcc atcactcacctccagcctcttctgct gtggccttgattcagcctctcacaat ttttttttaaccagaattctggcagt ggcccctgacttgcctctgtgctccc agccccgctgtcctctgatccatcct ccatgccagcctttttcaatctgctg gtcacgattcattgatgggttaggaa atcaatggcatcacaactagcattta gaaaaaggaaataggcgttcccgccg tggcacagcagaaataaatccgacta ggaaccataaggttgcgggttcaacc cctggccttgttcagtgggttaagga tccggcattgccgtgggctgttttgt aagtcacagacatggctctgatccgg cattgctgtggctctggcgtaggcct gcagcatcagctccaattagacccct atcctgggagcctccatatgctgcaa gtgcagccctaaaaaaaataaaaaaa taaaaaaaaataaataaaagaagtag acaaattgtatagaacaaccctgagt atgttgcctgagcacatataacaagg gtaagtattatttcaggaaactctgg tttcacagatactcttggcatatgga cccctagagtcctgatgtaaaatata ttcttcctgggatcttaggcaagaag tttgaaagctccaactctgcactgct gccaaagaaatgatttttaagtgcaa aactcttcccgttcccttccctgtat aaaattccataggatctctccagtgc ctctaggataaaggcagttttcattc tctagttcaaggtgagagaagatttt aattatttcacgttttagtggggaat tcaagagtctggcacctgacatttgc tgaactctctccattatccctctcta gttccccagacgcatcctatggtaga aattcgcaaactagagtgagcgtcag agtaacccaaggaaactgggtaaatg cagctccctgggctctaccccctgag attctgattcagtagatctgaagcag agccctggaatatgcatatgcatcat tgtgtcacaccaagcattctgggtaa tgagagttgatgttaggttctcagta gtaagacaagtatagagattccgggg gactgagtgctcagctctgccttggg gaggagggagagggctaaagagaaca ggagatggggacagggaatgctcaac ctccaatcttaggcatttgagctatg tcttaggggtcaggaggaggttacca atatagtgattaagagattgaggttc cagtcagagggatatgctggagaagg ggggtgaaaataatgtcataggtttg gtgagtgcagatactttgagtttttt aatatttttattgaaatatagttgat ttacaatgctcttagtgagtacaatt actttgaataagtgcatagatgtatg ccattcttccagaaatgatttattga gctcctttgggcatcatgctaagtac aggggaaacagctgtgaagaggtcct tcccttatgaagtcattcatcccctt cagtaaatgaaggtaaaggaaaagga tgagacagggacgccgtgttggacca gggtcagaaaggccttataagacctt gcctggagggcaaggaacttgcctgt gagtaaggagagcttgagaaagcgat aaagcaaagaaggaacattactgcat tgtgttttagaaaaaccatgtcctgg ggaagaactcctagagtcaggggggc cagttgggagactgtgcttttttcca ggaggagataagtgaggctgctggct gagatggagcaaggatttagagaagc agatatgagattcatttagaagttag acattttaggatctgacacataattt atcaccaaaaccagtgcatctctggc tttgggccaccagttttggagaagtg gaatgtagggacctaccattacctgc caatctttactacacagatgcctatt tccctcctcatatttcctttctccag atcacgtcctattctattgccaggac tcaagattccaccttgcatgcagtga tccatcttcacactggatggacagct ctagggatgtcagagcacactcttgt ccatactgctgactgggtctcctgtc agcccatctgtctatcagctgtggta ttattagtataataagagggctgtat atgagagacacaaaattctaggtgta gctcaaagataggctagagttattcc tatgtacaacaaatatttatgggacc ccttctgtgtactgtcatggttgctg ctttcatcatacttgtagtctaatgg aggtgggggcagggcaggaataagcg gatgtccacaaatcagtaagaccact tatattcaacattttcataatttagt tatttgagcccaaagggtccacatcc gtggtattccaacttttttttccccg gacatggatctttatctttttttttt tttcttttttgcggccagacctgcgg catatggaagttcccaggccaggggt tgaatgggagttgcagctgcctggtc tacaccacagccacagcaaggtggga tctgagctgcatctgtgacatacacc gcagctgaggtaacaccagattctga acccactgaatgaggccagggatgga acccgtctccttatgaacactatgtc atgttcttcaccctctgagccacaac gggaactccagacttcgtctttaaat gtattctgacttggagagctatcaca ctaagcaattaacaggagctgacctg gtttaggctggggtggggccctactc ctcaatgttccctgaggcacatctgt gggacccctgggcatcatctatctga gcagccttagagctgctcatccagtt gactgttgatgtagaagtgcaaactt ctgccttccttatttgttgctttctt ttttcattgttctctcccctttgtgt ctttaag CAAGGGCATCGTAGAGCCTCCAGAAG Exon 14 Seq ID No 23 GGACTAAGATTTACAAGGATTCCTGG GATTTTGGCCCATATTTGAATATCTT GAATGCTGCTATAGGAGATGAAATAT TTCGTCACTCATCCTGGATAAAAGAA TACTTCACTTGGGCTGGATTTAAGGA TTATAACCTGGTGGTCAGG gtatgctatgaagttattatttgttt Intron 14 Seq ID No 41 ttgttttcttgtattacagagctata tgaaaacctcttagtattccagttgg tttctcaataagcattcattgagcct tactgactgtcagacggagggcgtat tggactatgtgctgaaacaatccttt gttgaaaatgtagggaatgttgaaaa tgtagggaatgaaatgtagatccagc tctgtttctcttttggaggattcttt ttcctccatcaccgtgtcttggttct tgtttgttttgggtttttgtgggtgt tgtattgtgttgtgttggttatggca gtgacagctatttaaactgtgaaacg ggggagttcccgtcgtggcgcagtgg ttaacgaatccgactgggaaccatga ggttgcgggttcggtccctgcccttg ctcagtgggttaacgatccggcgttg ccgtgagctgtggtgtaggttgcaga cacggctcggatcccgcgttgctgtg gctctagcgtaggccagcggctacag ctccgattggacccctagcctgggaa cctccatatgccgcaggagcggccca aagaaatagcaaaaagacaaaataaa taaataaataaataagtaagtaaaat aaactgtgaaacggggagttcccttc atggctcagcagttaacaaacccagc taggatccatgaggatgtaggttcga tccctggccttgctcagtgggttaag aatccagcgttgctgtgagctgtgat gtaggtcgcagatgcagcccagatcc tgcattgctgtggctgtggcgtaggc tggcagctgaagctccgattcaaccc ctagcctgggaacatccatatgctgc aggtgtggccttaagaggcaaaaaaa taaaaaaataaaaaataaataaattg tgggacagacaggtggctccactgca gagctggtgtcctgtagcagcctgga agcaggtaaggtaaggactgcagctg ggtaaggactgaattgcaccaactgg gaagtaagcctagatctagaacttaa gttagccctgacatagacacacagag ctcaccagctaagtggttcagcttat aagctggtcactgaaactgaggatgt ccacaaaagcaaaataagtagcaaca ggcagcgggatgcaagagaaagagga ggcctaaaatggtctgggaatccctg ccatacctatattttatcctacttat atttagtgcctgaatgtgtgcctgga gagcaaagtttagggaaagcatcggg aaatgcacagtattcatacccttagg aacaaagatcagttacctccagggta aagactatttccaagtttaaatttca acccctgaacattagtactgggtacc aggcaacacttgccatcctcaaaatc aatgaatcctaaaattcaacctgggg gtcagtgacagtctgtgacaaagttt ttgctggtcagtaacgaaataagtat gagcaccatctgagtatggtcaccaa gatgtcaactctctttcctttggacg aattgtcattattccaagattaggtc ctttctatttttgaggtgtgaaaaca tctttcctttcataaaataaaaggat agtaggtggaagaattttttttgttt tttggtctttttgctatttctttggg ccgcttctgcagcatatggaggttcc caggccaggggtcgaatcggagcttt agccaccggcccacgccagagccaca gcaacacgggatccaagccgcatctg cagcctacaccacagctcacggcaat gccggatcgttaacccactgagcaag ggcagggaccgaacccgcaacctcat ggttcctagtcggattcgttaaccac tgcgccacgacgggaactcctaatga tactcttttatatttagctactatgt gatgatgagaaacagtccacatttta ttattttttagccaatttgatatctc attactaagataatgataattttctc tataaattttatttaagttagtgtta tgaagtggttttgctagtgtagaagg ctaggatttgaattcagttcaagaaa gaagagagggagggagggagagggat gggtagagggatggggcagtgggaga gagcaaagaggagagacagtttttgt attaattctgcttcattgctatcatt taagggcacttgggtcttgcacattc tagaatttctaaggaccttgaccgcc agattgatatgcttcttccctttacc atgttgtcatttgaacag ATGATTGAGACAGATGAGGACTTCAG Exon 15 Seq ID No 24 CCCTTTGCCTGGAGGATATGACTATT TGGTTGACTTTCTGGATTTATCCTTT CCAAAAGAAAGACCAAGCCGGGAACA TCCATATGAGGAA gtaagcaggaataccagtggaagtgc Intron 15 Seq ID No 42 ccctttcttccttccttcctaaataa acttttttattttggaacaactttag agttacagaaaagttgcaaagatatt atagacagtagtgtttatatatatat ataaatttttttttgctttttatgac cacacctgtggcatatggaggttccc agtctaggggttgaattggagctaca gctgccagtctgtgccataaccacag caatgcaggatctgggccacgtctgt gacctacaccaaagctcacagctgga ttcttaacccactgagcaaggccagg gattgaacctgcatcctcgtggttcc tagttggattcgtttccgctttgccg caatgggaactccaaattattgttaa tatcttactttactggggtacatttg ttacaaccaatactctgatactgaaa cattactgttaactccgtacttgctt ctttttgagtcatttgcaaagactgg cttcttgacctgcttccttccaaaca gctggcctgcctatgctgttctcaga cctgcaagcactgatctctgcccccc ttgccttctctccagtggtgtctcct tccccaaacaaacccagtgtggctct ggaaagggagttaagtcaacataaac caacacatattttgttgagctccaat tttgagcaaatccctcacctacggca gacaggcatgatgttaagaactaggg ctttggacacaaggtcaagaccaaga agggttcctcacccctactgattcag ataaccaataatgaggctttgaatcc ctgtccaaaggttgttttttttccct tctattgagcttcttgccaccttatc agttttttttatgacagtcaaatgac atgatatatgtgagcatacatggtaa tttttaattctatataaatgaatcac taaataaattaggaggatatatagtc cacctttaagcgtattacacgtgtca catgaatgtgtggcgacttaattgta gaggtttaaatgtagcttcctataat agatgtgttcctaaactacattttaa tcattggacttgtatttttatgttag cacttgctgttgaagaaaagcctatg ccaaaagttcagtgaaaccaataatc cactgccagctttctgagttaaaaaa aatccctgggttttcacacacaggaa caccctgtgtgaaacactcatttaga gcaaaatgcatctgataaggagttcc tgttgtgcctcaactggttaaggacc tgacattctccatgagaatgtgagtt tgatccccggccccactcgatgggtt aaggatctggtgttgccacaaactgc agctccgattcatctcctagcctaga aacttccacagcccagaatatgccac agaattcggctgtttaaaaaaaaaaa gaaaaaaaaaagaatcataaatgtgt tggtttgttcaccaaatacatgataa cttgctcttgccaagctcagcttcat aaatattaagtcatttaatacagcag ccaccttatgaacagatattactata cttcccatttacagataaggaaaatg ccatatttaaccaagagattaaataa ctttcccgaggtcttatagcaagtaa atcatggtgcaggggtttgaccacac gcagtctatcctccagagtctgtgta tttagccactgttttactttcaaatt taaatttataaaacttctaaattatc tgttaaccataatctttggaattttt aaaaccacgagttcctataaaatgtt tcattgaaagtaagtcacttttccat agcttttgataatacatctgtaggat aaagtaagccacagctctcttgcaga cttggtacaccctggggcaaagcatc atgcctgtcacgtacatggtggtcct tactttgactctcagtgcttttattg cccaggaattttgtgagatttctagt tgttgaggtttgtttaaagaggttat gccggtacttggaagagctcttttct tgctacctggagccttctcatatttc ctttttgaggagggacatgaattgcc tttcaaactcataaatatattttcta gtacacaagtctccatcttccttaga cgcatggctcctggagttctccatcc tcctgctccactttgggtgggctcct ctctgggtctgccaccaatctgccac ccagagacatccttgacccacttcca gaccccaccatggcttcactttcttc gctttcctcctttgtggaaccttctg cttaagaatctgaggaagaaaatttg cacgtgagctaaactggaggtacttt cctgcctggtcttgcacgatagcttg gctgagcccatgatgctgggtggctg ttactttccatggacacccgaaggcg ttgctcctttggcttctagttgcatg cagtgttgcttatcccaggctgatct ttcttccactgtaggtgacttttaag aattaagggattaatctatatctaca acaacaacaacaaagaccttttcaag ctgaggtagggctttctgtatatgtt tggagtggttatccagcagactttac ttgaaggcaggggtcatatcctcaag tgctcataaacggaccacagaaagat ctcataattgggtggagctgggtggg gaccgtgtcatgtggccaggaaatgc cagatgggaagggagtggcccttact gagctccagctgaactctgaattttc tagaaaactcagaaatctggattttt catgtgtaatacccagatttatagat gtggaaagctaattcttttttttttt aagggactataggcaatgaactaaga tctaggttgtatttggacaaggggtc atcagtttaagctgtgtagttgagcg ctcagctattgggctgagggacccct aaatactgagacggggaggtccttgc tctggggcatcacaagtacactccct ggtctcattcaaacacttttcctaca aaattgatcccatttcttcagtgcac tgtctgaatgcatttggcccagagcc gtgctgaggcatagggaaggggtcca cggtttcatggcatcgttttgtgctg tgtgtccctgctgtcgtccaggatac ctacctctcctcctcctgcatctgaa tgtccccccacagactctctgggatt ctacagcctctggcctgttcctcaga cacctcttacctgccagctttccaga ttcacattagttagtccaaatctact gccgtcagtgactcacttcatttctt cttctccgaggcagttcagcccggta cagttgttttgtcaacacttcagttg agtctggaagatgtgcatgggttatg cacgagagcggtccatcattttgagc tagaagtcctttctcagcccagagac aagtcctcatctcctttacttcctga ctcttcttcctctgcatccttccaag atatctctttctccagccaccaccta aatctcttcttttcccggggttccgt gctcaacccactcttcttcttaaatc tgtggctgggtgaacgcatctgctgg caccacttctctgctaaagactccaa aaatccataggtcctgcccggccttt gcccacctctctccaacactgtccag ctttagatgtagagctaatcccccca gagatatcattccctggatgtctaag tcctttggtatctcactttcagcgtg ttcaaaatcctcttacaactgttctt tctccttttccatcttgattattggc aacatgccagcctttcccctaccccc agcagtgagccaagctagaaacaagg gcttaatcttcaatctttccttctcc atccctaaacctaatgagtctccaag cccttcccagtttacaccctaaatgt tgctcaaaacatcccctagttcttcc acgtgctctcctctatattgaaaggt caagaaaggccatcttccctccactg tgaggaaatagatcttgatactgccc ctgagctgggcagtcctcgacctgac aaactgtgcagtgtttctaaatctct actggcaaaatgagagtgcctttgac ctgtgttgcgatctcagatcacagtg gatgtaattgttttataggaatggtg aacgaaaaagaagtaaatccctaatg ccaaactcctgatcattctatgtcat ttaatagcctgtcatttatgataaag tttcctctactggcattagcacaata cttctcaggaaaaaaaaatatgatgc cagatactgaaaagctcctgggtaaa catgaacatgggtaccgataaaatgg tgaagccagtccaatcttagagtgac ttcccttcatgctacttcatgctctt ttttttttttttttttaagaaaaacc ccttttttttttctcacaccagtcac agaggagaccgaggcttagcaaggtt aaggtcacatgattagtaagtgctgg gctgaaactcaaaaccatctctgctt gtctcctaaccctgtgcacctctgac tattcaacag ATCCTGTGTCAGGAGTTGGGATTCTT Exon 15a Seq ID No 25 TGAAG gtaagggccttgaccaccgaattaag Intron 15a Seq ID No 43 gtaatcttgctctgtggcaggccttg ttttcagtattttaagtacactggct caggtaatcctcacaacagccccagg aggaatgttctattacctccactgta tagatgaggaacttgaggcacagaat ggttgccaaggtcacacagctatatt gggggttcatacccagccatccaact ctgtctgtactctctgccactctgca cccccagctcctgatccacttcctgt ttccatccctcgatttctgctgcact caggggcccctctccccctcggcctg tgagatctgcttcagtaggcttttct ccctgactcctccatccctgtcctta caggcagctgcttctctccgggacac gaggggtccatacggacactctctac tggctgggttgcgcctaactcgtgat tcctcctctgtttcag ATTCGGAGCCGGGTTGATGTCATCAG Exon 16 Seq ID No 26 ACACGTGGTAAAGAATGGTCTGCTCT GGGATGACTTGTACATAGGATTCCAA ACCCGGCTTCAGCGGGATCCTGATAT ATACCATCATCT gtaagtccgaaaatgcctgtcgtgtg Intron 16 Seq ID No 44 tgccttaggctgctgcggaggaggcc agggctatataagcagagtcagtgac tgactgtgccctgcagtgttgatggc catggagattccaccgttagagcttt tttctttgttaaccttgaaggcaaat ctggttaggaagataactttcaaaga gtcaccatctggacattcatgcccat gtgcttcaatcctgtatacaagcagt ttagagtacagggaagggaaggacat tatgaaagggagagggtgtgtttgga tccagcagctccatcctcagaattta tctgaagacactgcaaaattactaag aatcactatgacaagaatgaggatgg ggtgatatggcaaagttgtgatcctg gaagaccttcatctcccatgttgccc aactctgaacatgaatttggtgaact agttggttaaggggatgatcctccaa gtttctccctggttgagctccaaaaa ccatgtaagtttctcatagcaaaacc gtataggtccttagggctttagttgg aatatttgtgctgaaatgctggaaag ccccatttgccatttttgtatttgca aaataatcatcaagaggggagaatgc attctttcatgaccactgaccctctg aaaaggtcaggaatttagtctgaagt aggcaagcctcctaccccgcttctgc catgagcttgcacgcacaggcctgtc ttgacatttcttctttatagatttct ttttgaatatcttgaaattgctttaa aaatatttaaagaatgtagaattata taaaataaaaaggaaataaccccaca cctcccacaaaaccctgtttcctgcc tttctccacccactctccagggtaac acttggtaacagcatagttgtatcac cccaggcctatttttgagcatatcag catttcaagaaatgtattttttctca ataaaacatcccttatagttgaggag gggaggttatcattcctgggttttgt tttttttttttttttaatgtaatcct ggtacatcggtaatttgcatttttta ttcattaatatctttggtatttctag tgttgggacacacaggtcaacctcag tttttgggtttttttttttgtctttt tgtctttctagggccacacctgcagc atatggacgttcccaagctaggagtc taatcagagctgtagccaccagccta cgtcatagccatagcaacgtcagatc caagccgtgtctgtgacctacaagca cagctcatggcaacaccggatcctta accactgaacgaggccaggggatcga acacacatcctcatggatcctagtca tgttcattaaccactgagtcatgatg ggaactccaacttcaactattttaat gtctgtaaaacattccatttggaaac catttcatttgtaaagcaaaatgaaa acattttgttcattttcaacagagtt cgtagctgacttctgttctggaaaaa aggaaatggagcaaatttgagtgaga aagattcaaagataacttttctttta aaaaaaattatatcttggaaacttct gggctattgattctgaagactatttt tctatatactgttttgatagcaaagt tcataaatgtgaaaggatcctgcgat gaatcttgggaagcagtcatagccca atatatctttgttgcttttaaaatga gatttagtttactaaatatttttctg atcataaaaataacacagatctaccg cagaaaatttggaaaaaaaaaaactt ttaaattcaaaaaacagttaaaccac aaatgatcccaccatccagagagcaa tttgtactttggtgtctagttcatct ttctttttctgtttacaagcacatat accacaagcattttttcaaaaaatga aaatgggataatactatacatacgtc tgtacacctgcatagttactgaacag tctttgatctaccctgtaagtttcta acttttcattatttgaaatgatgttt tggcaaagaaatatgtaggtgtgtct cgcacactttcataatgatttcttag gataaatttcttaggataaattcata atgatttcttataataatccatactc tgccaactgatcttcagggaagccaa ctcgccttctcagaaataacatataa cccatttacttgccctctcaccaata ctaggtcctaatgtttttgtgtacag attctatatttttacatacaagaatt ccttaaagcaaggcatgtcacagaaa aatagaaggaagacacaattgtcatg tttaaggactgcattctgtaccaaaa atgctaagttaaatgaacatctgaaa cagtacagaaacgctatctttcaggg aaagctgagtaccaggtactgaacag attttggcaaatacagcaggcatgga tgtttccaaaacatgtttttctactt tatctcttacag GTTTTGGAATCATTTTCAAATAAAAC Exon 17 Seq ID No 27 TCCCCCTCACACCACCTGACTGGAAG TCCTTCCTGATGTGCTCTGGGTAGAG AGGACCTGAGCTGTCCCAG gtaaagcatcctgcaggtctgggaga Intron 17 Seq ID No 45 cactcttattctccagcccatcacac tgtgtttggcatcagaattaagcagg cactatgcctatcagaaaacctgact tttgggggaatgaaagaagctaacat tacaagaatgtctgtgtttaaaaata agtcaataagggagttcccatcgtgg ctcagtggtaacgaaccctactagta tccattgaggacacaggttcaatatc tggcctcactcagtcggctaaggatc cagtgatgccgtgagctgcagtgtag gccacagacgtggctcagatctggtg ctgctgtggctatggtgtaggccggc cccctgtaactccaattcgaccccta ggctgggaacctaaaaagaccccaaa aaagtcgctttaatgaatagtgaata catccagcccaaagtccacagactct ttggtctggttgtggcaaacatacag ccagttaacaaacaagacaaaaatta tcctaggtggtcagtgggggttcaga gctgaatcctgaacactggaaggaaa acagcaaccaaatccaaatactgtat ggttttgcttatatgtagaatctaaa ttcaaagcaaatgagcaaaccaattg aaacagttatggaagacaagcaggtg gttgtcaggggggagataaggggagg caggaaagacctgggcgagggagatt aagaggtaccaactttcagttgcaaa acaaatgagtcaccagtatgaaatgt gcaatgtgggaaatacaggccataac tttataatctcttttttttttttgtc ttttttgccttttctaaggctgctcc cgtggcatatggaggttcccaggcta ggagtccaaacagagctgtagctgcc agcctacaccagagccacagcaacac gggaaccttaacccgctgagcaaggc cagggatcgaacccgagtcctcacag atgccagtagggttcattaaccactg agccacgacaggaattccagggtctg ttgtgttcttaaaacacttccaggag agtgagtggtatgtcataagtaaaca ataaatgttaaccacaacaagcttat gaaataaacaggaaagccatatgacc tacaatcagtcattgggagaatccac aaaaggttgagcagaggatcaattcc agctcacactccagttttagattctc ccctgccttaaagcatcacagactac ataatctgagctgaagaataaaaatt aaaactcaccccagtgcaaaacagaa atgaaaaagtattaaaacgaggttca tactgttgttcattagcaatatcttt tattcacag GGGTGCCCAACAACATGAAAAAATCA Exon 18 Seq ID No 28 AGAATTTATTGCTGCTACGTCAAAGC TTATACCAGAGATTATGCCTTATAGA CATTAGCAATGGATAATTATATGTTG CACTTGTGAAATGTGCACATATCCTG TTTATGAATCACCACATAGCCAGATT ATCAATATTTTACTTATTTCGTAAAA AATCCACAATTTTCCATAACAGAATC AACGTGTGCAATAGGAACAAGATTGC TATGGAAAACGAGGGTAACAGGAGGA GATATTAATCCAAGCATAGAAGAAAT AGACAAATGAGGGGCCATAAGGGGAA TATAGGGAAGAGAAAAAAATTAAGAT GGAATTTTAAAAGGAGAATGTAAAAA ATAGATATTTGTTCCTTAATAGGTTG ATTCCTCAAATAGAGCCCATGAATAT AATCAAATAGGAAGGGTTCATGACTG TTTTCAATTTTTCAAAAAGCTTTGTT GAAATCATAGACTTGCAAAACAAGGC TGTAGAGGCCACCCTAAAATGGAAAA TTTCACTGGGACTGAAATTATTTTGA TTCAATGACAAAATTTGTTATTTACT GCGGATTATAAACTCTAACAAATAGC GATCTCTTTGCTTCATAAAAACATAA ACACTAGCTAGTAATAAAATGAGTTC TGCAG

TABLE 10 Genomic Sequence of CMP-Neu5Ac Hydroxylase gene ctgccagcctaagccacagccacagcaacgctgggtc Seq ID No. 46 tgagccatgtctgcagcctatgccagagctccccgca gcgccggatgcttaacccactgagcaaggccagggat tgaaccctcgtcctcatggatagcagttgagttgttt ccacggaactcttaggggaactcctgattatttttta tttaaatttatatttctctgactttttcgtgtgctca tcagccactgactgtgtatctccattagtcatggttt gttaactctgtcattcaaaccctcttcatccttgcta cgcagataacatcattataataaaatcgtgcctgaag accagtgacgcccccaagctaagttactgcttcccct ggggggaaaaagaagcaccgcgcgggcgctgacacga agtccgggcagaggaagacggggcagaggaagacggg ggagcagtgggagcagcgggcagggcgcgggaagcac tggggatgttccgcgttggcaggagggtgttgggcga gctcccggtgatgcaggggggaggagccttttccgaa gtagcgggacaagagccacgggaaggaactgttctga gttcccagtCCCGACGTCCTGGCAGCGCCCAGGCACT GTTATTGGTGCCTCCTGTGTCCACGCGCTTCCCGGCC AGGCAGCCCTGGCGGATCCTATTTTCTGTTCCCCCGA TTCTGGTACCTCTCCCTCCCGCCCTCGGTGCGCAGCC GTCCTCCTGCAGTGCCTGCTCCTCCAGGGGCGAAACC GATCAGGGATCAGGCCACCCGCCTCCTGAACATCCCT CCTTAGTTCCCACAGgtgagaaggcttcgccgctgct gccgctggcgccggcagcgccctccacgcacttcgta gtgggcgcgcgccctcctgcattgtttctaaaagatt tttttttatccgcttatgctatcagttactgaggaag tatttacaaatctactattattttgaatttgcctttt tctccttatagtttatcagtatctcttgagactgtta ttggtgcctgcaaatttaaaatgattggggttttatg aggaagtgaaccttttatctttatgaaacgcctaact gaggcaatgttaattgcttaaaatactttctttatta tcagtgtggccatgccagtgtcctcttggttagaatt tgcctgat............................. ............ctgccaaagctgggagatgggggaa agtagagtgggttattgaaactgaatatagagttcag catctaaaagcgaggtagtagaggaggaagctgtgtc aacggaaatactgagctgggttcacatcctctttctc cacacagTCTAATGCCTTGTGGAAGCAAATGAGCCAC AGAAGCTGAAGGAAAAACCACCATTCTTTCTTAATAC CTGGAGAGAGGCAACGACAGACTATGAGCAGgcaagt gagagggggctttagctgtcagggaaggcggagataa acccttgatgggtaggatggccattgaaaggagggga gaaatttgccccagcaggtagccaccaagcttgggga cttggagggagggctttcaaacgtattttcataaaaa agacctgtggagctgtcaatgctcagggattctctct taaaatctaacagtattaatctgctaaaacatttgcc ttttcatagCATCGAACAAACGACGGAGATCCTGTTG TGCCTCTCACCTGCCGAAGCTGCCAATCTCAAGGAAG GAATCAATTTTGTTCGAAATAAGAGCACTGGCAAGGA TTACATCTTATTTAAGAATAAGAGCCGCCTGAAGGCA TGTAAGAACATGTGCAAGCACCAAGGAGGCCTCTTCA TTAAAGACATTGAGGATCTAAATGGAAGgtactgaga atcctttgctttctccctggcgatcctttctcccaat taggtttggcaggaaatgtgctcattgagaaatttta aatgatccaatcaacatgctatttcccccagcacatg cctaactttttcttaagctcctttacggcagctctct gattttgatttatgaccttgacttaatttcccatcct ctctgaagaactattgtttaaaatgtattcctagttg ataaacagtgaaacttctaaggcacatgtgtgtgtgt gtgtgtgtgtgtgtgtgtttaccagcttttatattca aagactcaagcctcttttggatttcctttcctgctct ctcagaagtgtgtgtgtgaggtgagtgcttgtccaaa cactgccctagaacagagagactttccctgatgaaaa cccgaaaaatggcagagctctagctgcacctggcctc aacagcggctcttctgatcatttcttggaagaacgag tgctggtaccccttttccccagccccttgattaaacc tgcatatcgcttgcctccccatctcaggagcaattct aggagggagggtgggctttcttttcaggattgacaaa gctacccagcttgcaaaccagggggatctgggggggg ggtttgcacctgatgctcccccactgataatgaatga gggattgaccccatcttttcaagctttgcttcagcct aacttgactctcgtagtgtttcagccgtttccatatt aggccttccaccgtgtcgtgtcgtcaatcttatttct caggtcatctgtgggcagtttagtgcgaatggactca gaggtaactggtagctgtccaagagctccctgctcta actgtatagaagatcaccacccaagtctggaatcttc ttacactggcccacagacttgcatcactgcatactta gcttcagggcccagctcccaggttaagtgctgtcata cctgtagcttgcttggctctgcagatagggttgctag attaggcaaatagagggtgcccagtcaaatttgcatt tcagataaacaacgaatatatttttagttagatatgt ttcaggcactgcatgggacatacttttggtaggcagc ctactctggaagaacctcttggttgtttgctgacaga ctgcttttgagtcccttgcatcttctgggtggtttca agttagggagacctcagccataggttgttctgtcacc aagaagcttctgcaagcacgtgcaggccttgaggtct tccgacttgtggcccggggactctgctttttctctgt ccttttttctccttagtgggccatgtcctgtggtgtg tcttagccagttgtttaagggagtgttgcagctttat gattaagagcatggtctttccttgcaaactgcttggt ttagaagcctggctccaccacttagcggctctgtgac ctcggacacatttcttagcctttctgggcctcgctct tcttcctcataaagtgaaaatgaaagtagacaaagcc ttctctgtctggctactgagaggatggagtgatttca tacacataaagcacttaaaataatgtctggcatatga tacatgctcaataaatgtcacttacatttgctattat tattactctgccatgatcttgtgtagcttaagaacag aggtctttacaggaattcaggctgttcttgaatctgg cttgctcagcttaatatggtaattgctttgccacaga ctggtcttcctctccttcacccaaagccttagggggt gaacgatcccagtttcaacctattctgttggcaggct aacatggagatggcaccatcttagctctgctgcaggt ggggagccagattcacccagctttgctcccagataca gctccccaagcatttatatgctgaaactccatcccag agcagtctacatggtacactcccccatccatctctcc aaatttggctgcttctacttaggctctctgtgcagca attcacctgaaatatctcttccacgatacagtcaagg gcagtgacctacctgttccaccttcccttcctcagcc atttttcttctttgtacataatcaagatcaggaactc tcataagctgtggtcctcattttgtcaatctaatttc acagcctcttggcacatgaagctgtcctctctctcct ttctgcctactgcccatgagcagttgtgacactgcca catttctcctttaacgacccagcctgctgaatagctg catttggaatgttttcaatttttgttaatttatttat ttcatcttttttttttttttttttttttttttttttt agggccgcacccatgggatatggaggttcccaggcta gggatccaatgggagctgtagctgctggcctacacca cagccacagcaatgcacaattcgagccaatctttgac ctacaccagagctcacggcaacactggattcttaacc cactgattgaggccagggatcaaactctcgtcctcat agatacgagtcagattcgttaacctctgagccatgat agttgttagttactcattgatgagaaaggaagtgtca caaaatatcctccataagtcgaagtttgaatatgttt tctgccttgttactagaaaagagcattaaaaattctt gattggaatgaagcttggaaaaaatcagcatagttta ctgatatataagtgaaaatagaccttgttagtttaaa ccatctgatatttctggtggaagacatatttgtctgt aaaaaaaaaaaatcttgaacctgtttaaaaaaaaaac ttgactggaaacactaccaaaatatgggagttcctac tgggacacagcagaaatgaatctaactagtatccatg aggacacaggtttgatgcctggcctcgctaagtgggt taaggatatggtgttgctgcagctccaattcaacccc tatcctgggaacccccatatgccaccctaaaaagcaa aaagaaaggtgctgccctaaaaagcaaaaagaaagaa agaaagacagccagacagactaccaaatatggagagg aaatggaacttttaggccctatctccaactatcacat ccctatcaccgtctggtaagaaatggaaaaaatatta ctaagcctcctttgttgctacaattaatctgattctc attctgaagcagtgttgccagagttaacaaataaaaa tgcaaagctgggtagttaaatttgaattacagataaa caaattttcagtatatgttcaatatcgtgtaagacgt tttaaaataattttttatttatctgaaatttatattt ttcctgtattttatctggcaaccatgatcagaaatct ttaaacaatcaggaagtcttttttcttagacaaatga aaatttgagttgatcttaggtttagtacactatacta ggggccaagggttatagtgtgactattaaatcacaga taatctttattactacattatttccttatactggccc cacttggatcttacccagcttagcttttgtatgagag tcatccttaaagatgactttattctttaaaaaaaaaa acaaattttaagggctgcacccatagcatatagaagt tcctaggctagcggtcaaattagagctgcagctgcca gcctatgccacagccacagcaatgccagatctgagct gcatctgtgacctacactgcagcttgcagcaatgctg gatccttaacccattgaacaatgccagggattgaaca cacatcctcatggatactgctcaggttcctaacctgc tgagccacagttggaactccaaagcagactttattct gatggctctgctgatctctaacacgttattttgtgcc atggtgtttatcttcactttactcaagtcagggaaac acgaagagtctcatacaggataaacccaaggagaaat gtgcaaagtcacatacaaatcaaactgacaaaaatca aatacaaggaaaaaatatcttcactttcaaaatcacc tactgatgatgagtttatatttccttggatatttgaa tattagctatttttttcctttcatgagttttgtgttc aaccaactacagtcgtttactttgatcacagaataat gcatttaagccttaaatagattaatatttattttcac catttcataaacctaagtacaatttccatccagGTCT GTTAAATGCACAAAACACAACTGGAAGTTAGATGTAA GCAGCATGAAGTATATCAATCCTCCTGGAAGCTTCTG TCAAGACGAACTGGgtaaataccatcaatactgatca atgttttctgctgttactgtcattggggtccctcttg tcaacttgtttccaatctcattagaagccttggatgc attctgattttaaactgaggtattttaaaagtaacca tcactgaaaattctaggcaagttttctctaaaaaatc ccttcattcattcatttgttcagtaagtatttgatga gaccttaccatgtgtaaacattgcactaggtattaag aaatacaaagatggataagatagagtcggcgtaaatg agatgatataatgagacgttataatgaaactcacaat tccagttgggaaataaagtccttcaaattccatgact ctttctggcacacgttagaggctacagcttctgtgtg attctcatgctggctccacttccactttttccttctt cctactcaagaaagcctatagaaatatgagtaagaag ggcttaatcataggaataaatttgtctctgttctaag tgattaaaaatgtctttatcagtataaaaagttactt gggaagattcttaaaactgcttttacacactgttcta gaatgactgttatataaataaaaaagtagatttgatc taacacaattaaatgacctttggaaatattgactaat tctcaccttgcccctcaaagggatgcctgaaccattt ccttcttttgccagaaagcccccaccctttgtctgtt gacctagcctaggaaatcttcagatcacgttgttagc acgaactggttacatgtgctgtacaaatactatttaa ttcatctgattaaaaaaaaagagataagaagcaaaag tttgactatcttaaactgtttgcgtaggtgagaggac aattgaccatctactttatgagtatgtaacccagaaa cttaaagctccttaagggagctaagtcttttggataa gacctatagtgagaccttttagcaaaatggttaagac tgaatggagctcactagcgtgggttcatatcctgatg ctcaaacacgcaattaaatgactttaggtgggttagt ctctgttccttagtttcctcaatgggagataatattg gtagtagcgattttactgggttgttgaaagaacatct gttaaatgttcagaacgtgttacgacagagtacagag taatgatttgcttgtatatgtatgactcaaatagtct gccatatgccttgtgactgggtcctgtggagcaggaa ggagggatttcccacccagcagaaagttgggtaaact ggaaaatagactgaggccaggaaatgatgcaaagcgt tgatgttcactgccacggcaggtgaagggcagggcca gagttgtcagtagggtcaggggaggactggaaataac caagacccactgcacttttcagcctttgctccagtaa ggtaatgttgtgagagtagaaaattttgttaacagaa cccacttttcagtacagtgctaccaatactgtagtga tttcataccacatcccaagaaagaaaaagatggctca atcccatgtgagctgagattatttggttttattgtta aataaatagcattgtgtggtcatcattaaaaaaggta gatgttaggaaagtagaaggaagaagactctcaccta cattttcatcactgttttggtatctgccagttgtcac cttggtccccttccccgcctctcccctgcctcctctt cctccttctcctttttttggaatacaattcaggtacc ataaaatttacccttttagagtgtttgactcaatggt ttttagtattttcacatgttgtgctattactatcact atataattccaggtcattcacatcaccccccaaagaa accttctaactattagcagtccattcccttcttccct cagcccctggcaaccactaatctacttactgtctcca tggatgttcctatattgaatcaagctagcataaaccc cacttgctcatggtcataattcttttttatagtgcta aattacatttgctaatattcaattaaggatttctatg tccatattcataaggaatattggtgtgtagttttctc tttgtgtgatatctttgtctggttgggggatcagagt aataattactgctctcatagaatgaattgagaagtgt tccctccttttctatttattggaagagtttgtgaagt atattggtattgattcttctttaaacatttggtcaga ttcaccagtgaagccatctgggccatggctaatcttt gtgaaaagttttttgattactaattaaatctctttaa tttgttatgggtctgctcctcagacgttctagttctt cttgagtcagttttgttcatttgtttcttcctaggac tttctccctttcatttggattatttagattgatagta atatcccccttttaattcctggctgtagtaatttggg tcttttctcttttttcttggtcagtttagctaaaggt ttgtaattgtattaatcttttcaaataactaactttt ttgttttgtttgttttttgttttttgttttttgtttt ttgtttttttttgctttttaaggctgcacctgaggca tatggaagttctcaggctagaggtctaatcggagcta cagctgctggcctataccacaaccatagcaatgccag attcaagctgcatctgcgacctacaccacaactcggc cagggatcacacccgcaacctcatggttcctagtcgg atttgttaaccactgtgccacgacgggaactcccgcc cattttttttaacacctcatactttaacataaagatg ggcttcacatggactgatagctcaaatgaggaaggta agactatgaaagtaatggaagaaatgtagactatttt tgtgacctagagattactgatacttcttgacttttca aacaatacttcaaaagtacagcccaaagggaaaaaag aaagaaaaaagaaacacacatatacacaaacctagtg aataagatatcatcgatacactacagatttctatgaa ctggaagaccccatggacaaagttaaagaacatatga tagtttgagtgattattttgcaatatttacaaccaat gagggaatattatccagcttataggaggaagtaatgc aaatcgacaagaaaaagataggaaacccaatataaaa attaagaaaatacaaaaattaagaaaggatatgaact agcattttacaaaagaaaaatctccaaaagtcaatca gcacatgaaaatatgctcaaacctattaattattaga aaactacagactgaagcaatgaggtgctttactttac atctttttgactgataaaaagttagaaacaaaggtga tatcaaatgtcagggataaaaggatatagaaatcgtc atgcctgtggtgggagtatggccggtgcagtcatgtg ggaaggtaatctgacagtggttaggcagagcaggttt atgaatacactgtggcccatcaatcccacgcctgttt atgtaccaaagaaatcctgttgtggcagaatctatgg gtccacccctgggagcatgaattaataaaatgtggca ccagggtgtgtgaaactccagctagagatgagatgtc cacatggcaacatgaatgcatcttagaaacatagatt tgagtgaaaaagagtaagaaacagccgggaaacccaa taccatttataaaaattaaagatgcacacatacaatg tagtaaatattttgcatgaactttcaaatggttgcct acagggggggagagtaaagaagagtagaaaacaaaga taaagggagtaagtaagtagctctgcctggactgaat ataatgtgtcatgaactgagaaatatggttaacataa tcctcttaacttgaggtcctaaatgaatgaatgagtc cactattcatttacccattctttaatgtgtattgcat tataatccatttttttagaaccaacgaattttgttcc cataactactaatcagcctgccttttctccctcattc ccttatcagctcaggggcattcctagtttttcaaacg ttcctcatttgaaccaaaaatagcatcattgtttaaa ttatacttgttttcaaatacgatgcttatatattcca agtgtgtttgcccattttcttaggtggtagaaatttt tcattctacttttctatctactcagattttcccgttg gaattatttccattgctattaaacttagaagtccccc ctgtgatatgccatttttttcatactttttaagcact tggttgcttttctttgtgtctttaagcacctagaata cttataaccattgcacagcactgtgtatcaggcagcc cttcctcttccactaatttatggtccttctcttagac tatattaaactgttatttaattaggatcctctcttcg tccttatgatttaattattatagttttctaatatgtt tttattataattcctcttcattattcctccctattaa aaattttaatgaattccatttgtttgttcttctagtt aaatattaagtcataatccaaataacttagatgtcat tagtttatgtggtcaaagtaaggataccacatcttta tagatgcaggcagttggcagatgtcatgattttcttc agtgcataaatgcaatttatctttgagcaaggggcat aaaaacttttatggtattggctttgaaataatagtta agaactgcagactcagtttttcctgcttttcttgaaa aagaacacttctaaagaaggaaaatccttaagcatgg atatcgatgtaattttctgaaagtctcctgtaattcc ttgggatttttgttgttgtttgttggtcggttttttt gggtttttgtttgtttgttttgttttgttttgttttg cttttagggctgcacctgtggcatatggaagttccca ggctaggggtccaactggagctacagctgccagccta ctccacagccacagcaacatgggatcctagctgcatc tgtgacctaaccacagctcttggtaatgccagattgt taacccactgagcaatgccagagatcgaatctgcctc ctcatggacactagtcagattagtttctgctgagcca caatgggaattcccaattccttgtatttttgaactgg ttatgtgctagcatataattttgtttcttgaatcttt gtgggtttttttttttttttttttttgtctcttgtct ttttaaggctgcacccacagcatatggaggttcccag gctagaggtcaaattggagctacagctgccagcctac acaacaactgcagcaaagtggggcccaacttatatga cagttcgtggcaatgccggattcctaacccactgagc agggccagggatcgaacctgagtttccagtcagtttc gttaaccactgagccatgatagtaactcctgtttgtt cagtcttgaacctcctttttaattctttattccttga gggtgaaataattgccataataatactatcatttatt acatgccttctctgtgctaggcatagtgacactttag gatttattatatcacttaatccctacaacaactctgc aaagtatgtatcataatcctatttgacagatcaggaa attgcagcccaggatgcagataatatgcatccatcac aagtgactagatatagtccctctgctattcagcaggg tctcattgcctttccattccaaatgcaatagtttgca tctattgtatatgtgttttggggtttttttgtctttt tttttttttttgtcttttctggggcctcacccttggc ataggtaggttcccaggctaggggtcaaattgaagct gcagctgccagcctacaccacagccacagcaactcgg gatctgagcctcatctgcaacctacaccaaagctcac ggcaacaccggatccttaacccactgagtgaggccag agatcaaaccggcaacctcatggttcctagtcggatt cattaaccactgagccacgatgggaactccctaaatg caatagtttgctctattaaccccaaactcccagtcca tcccactccctcctcctccctcttggcaaccacaagt ctgttctccatgtccatgattttcttttctggggaaa gtttcatttgtgccatttttcattttacgggtaattt ttacttcagtttcttccactagcagttgtcttaaagt gagtataattaatattcatttggaaaatgtaagcaaa acattttttaaagggccatgcccacagcatatgaaag tttctgggccaggggttgaatccaggctccaagttgc agctgtgccctacactgcagctgggcaatgctggatc ctttaacccactgtgcccggctagggatcaaacctgc atttccacagctacccgagccattgcagttggattct taacccactgcactacagtgggaactcccacaaaaca ttttttaatgtcctttgaataaagtaggaaagtgctc gtctttgagggcagggcggcaatgccatttccacaag gtttgctttggcttgggacctcatctgctgtcattta gtaatgaataaaattgctgacagtaataggattaact gtgtgtggagatagccagggttagagataaaaacact ggagaagtcaaataagttgctcgaggtcctctagcta ataagctattaagtgggagagtgagggctagaaacag gccatctgtctcccaagcacatgtccattagtggttt gctgatagccttccagaacaacagagaggactctcaa acatggtcttgcctccctccaattgatcccctccatg tgcctcacagcgggtctttctaaaattaagttctgat tttaattctcccttgctatagcacttaggtatggctt tcagccgtgcaataaaaagcaggcaagagtggctcaa tcatataggaggttgtttttcttagatcccaagcagg taatcctgggcattatggttgttctgcgtttatcaag gagccaaattctctatcacctcctgttctatcctcct cagtatctggctctattcttcagcatctcaagatggc ttgtgctcctccaagcatggcagtcaaattccacaca agagggggaaatatgaagggcagacagtgctggtctc ctgagctgtccctctttgtcggggaaataaatgtatt ccttcatgtcccgtgagacttctgaagtagacgtctg cttacgtctcacccaccagaactatgtaaactgcaca tagtgctaggtctacatagccactcataactgccagg gggtgggaaatctttaaataggtgtaccaccacacaa ttaggatgctaatagtaagggagaaggagagaatagg ttttgcgcaagccaccagcatgcctgccacaattgct taaaattcttcattgacccctcattgccacaggatga aatccaaacgccttcttagttgggaatctgacctacc tgtctctcccacctggttcagacaccattctccttgg tcataaaattccagtcatttgtgaacatccagctccc ccatgcctccatgcctttgcacatgctgttcttttat cttttatgttgtccttttatcttttatccaaaagaga tatcccatcatcacatctcttttgtcagcccccaaat actttgtctttcaagttcagctggaggattacctcct atttgaaatcagctttgtctcttacaaccaaacaagg ttttccttccgagacactcccacagcaccttgaactc atctctatcaatcattcatttgattgtaatgaagttg ttggtggtatgcctgtgtctctgacacatctgcgatc tcatgagttccttaagtggaatgtgaatagcgggatg aacagtattggtcttcagccctcatctctgcagatgt tgcttgacccaaatgagcgttgccttttattttgatt ttgctttgatttgtctactccatgtacttgagccatg catttctgtcttagcgatgctttttaaaagtcatttt ttggttgattatccagatttgtccacctttgcttcta gTTGTAGAAAAGGATGAAGAAAATGGAGTTTTGCTTC TAGAACTAAATCCTCCTAACCCGTGGGATTCAGAACC CAGATCTCCTGAAGATTTGGCATTTGGGGAAGTGCAG gtaaggaaatgttaaattgcaatattcttaaaaacac aaataaagctaacatatcaatttatatatatatatat atatatatttttttttttttttacatcttatattacc ttgagtattcttggaagtggctagttaggacatataa taaagttattctgaagtctttttttttctttttccat ggtgagcagtggcttgatgtggatctcagctcccaga cgaggcactgaacctgagccgcagtggtgaaagcacc aagttctagccactagaccaccagggaactccctatt ctaaattcttgagcacattatttaggaacctcaggaa cttggcaggattacaggaaatatatctagatttaaaa aaaaatcttttaacagaggtcccaaaggagagtcatg cacagctatgggaggaagttcagaaactgcccttgct accagatcactgtcagataaaatggccagctacatgt ttctgcacattgccctaagatctttacaaacttttct gtgcatttttccacttttaaaagaaaatttcggggtt cctgttgttgctcagtggttaacgaacccaactagta tccatggggacaggggttcgagccctggcctcactca gtgggttaagaatctggcattgctgtggctgtggcgt aggctggcggctacagctcagattggacccctagcct gagaacctccatatgccgcaggtatggccctaaaaaa aaaaaaaaagagagagagagaatttcctccagaaaaa acactttggtagtttgggagaagtaaacaaccaaaaa ttaatttttctggagtattcgggaagcttgtaaaaat gggctcttacttttttgaggagacaaatgggaaccta cccagaagaggcacaatcacctgcatttgatttcttg acctctccctaccttctttgctggctttccacatttg gatttctgtgaccttatctctgctccttggtgttttc atttttcctgtggacgtgccagactatgggaagggag taaggcgttgatttagaatcctgtagtctctgcctgt ctctagtcattgttttcacccttctcaaaggaccttg acatcctgagtgagtccgcaagtaatttaggggagaa gccttagaagccagtgcagccaggctacatgactgtg tccacccactggaaccagtcatttttatacctattca cagcccccctaccatttaaatccccagaggtctgcca taacatctgtaactccctttcctggtaaattgtgttc taaaagactggtaacaaaagatattctgtggtacaga gcataattaaatacctgggagctgatttgagtggggt aaatcaactggtttgacccctaaaacccaccatgagc atttctgttctaataaagtaatgcccgtgctgggaat tgtgttctacggaaatgctcctgctgtgtctttcttg agtcctgtgtcattgaacatgcttaggagcaaaggtc ccccatgtggcttgtctgctaaccagcccagttcctt gttctggctggtaatgatccgatcatctgaatctcac tgtcttccaacagATCACGTACCTTACTCACGCCTGC ATGGACCTCAAGCTGGGGGACAAGAGAATGGTGTTCG ACCCTTGGTTAATCGGTCCTGCTTTTGCGCGAGGATG GTGGTTACTACACGAGCCTCCATCTGATTGGCTGGAG AGGCTGAGCCGCGCAGACTTAATTTACATCAGTCACA TGCACTCAGACCACCTGAGgtaaggaagggtgagccc tcaactccgaagaaaatgctgcaataaaagcactgtt ggttttcagctttttttgtaatcactgctcattctga ggtagattcgcttgggctgataaaaagagaactaatt cagataaatgcttgcatttgcatagcctcttttttta aaaactttttttttttttttttttttttggcttttca gggctgaacctgtggcatatggaggttcccaggctag gggtcgaatcagagctgtagccccgggcctatgccac tgccatagcaacatgcatagcctcctttttaaagtgc cttcctgttttataccattgggatgtgagaagagcta ttgtggaaangagcatggggtnataaccctggacctc tcacgtcctaccctcaggntagtgggaaaactctgag tttaaggacatcaaagtgactcctttttagttacatt atggnggaatcagcncatatttttacaaggggcggag ngtaanctgttggagtttacaagacatatggtggcat tgcaactacttaaccctactattatagcacaaaagca gccatagtcggtcctgaaggagcctgatgccttcagc tttataggcaatgacgtgtgaatatcacaaacagttt cctgtgtcaccaaacatgattgccttttgatttccct ttcaaccctttaaaaaaaggtaaaagcccttcttagc attcagcagcaggtcgctgtgttttgccaactcctga tctgtagcatttcgacaacactgagctctcaactttt gaaccctgagtccaccacatccttcagtgaaaccaga gccatgtgatactaaggatagaaacggaaacttcctg aatccaggcgatcaaataggagggagaaagaggaact ttcattgacaaaaccacaaatattgtgaatggactgt tacaaatattgtgaatgctcctattcccaaccccctg gcttcattacagggtcctatgtgttcatccttattga gaaatttgtattgctactgccaggttgccaataccca gcggtgcccatggtgttctaaaatgaagcaatttcaa ctttatttttttttcctgtgactttacatgacaagtt cacatgaaggatatactttgatagtaatgtccatggt tagggaatatacattgtttgctggttgactggcccct ggatttttctattgaaagtccatgagatctcgaaggc acaggtgtgttctctcgctttttaaggaaagggttta aaaacttaagtaattaacagctttagtaacaaattac ctataacacacttaaaaaccgaataccacccactgga gtattgtgctacgattaaaaatctacttgtctactac atgatatctttgtcccacagaaggttctggaaccaaa cttgtaatttcaggattatgagagccctgagttcacg cattgtgtaataactatgttgtgtggtagtcaatttg tacagcttgcttagagagaacaatgtcaagttaagga ggcgattgctttatagtgcctgtcacaagatgccatt gccattgtcctagcaagagatattctatgggagtata ctacattttagtgaggataagaactttttatggcatt tagtccggtcatttcccaaccactgtcctgaaaacca atttcattttgatttcaggggcttgtgtgggcaaagt tgccaggcattaaaaagccacttctcaactgtagtat cacaatgctttagttgggtagtgtattgcagatagct tatggctgaaaagttaccaagccttgcagttttcact cctttgagtttatttccttgacagaattgaccctgag ttttttgactcttacctgctcaactaataaacaccag agtcatttatctccattgctcttgtctgacctttatt taccgaataatgccttatgggttcacaaaaacaaggg gggagggggccagcatgccttagaaactgtctttagt caagaaatgngattttattatgtaaatatatgagtat tataatagatagtgttattaatagacaccagcaagaa ttgtcaataatttaaaaatcacaaattaaaatacatc catgttagnatcatttatcctaactcccaaagccctt taaagtggaagatttagatgttaacccagagattaaa gacatgttcaaagaatccttgatttttttttgaatcc cttgtttttagagaagaaaacctaatgattttccccc tctggattctacatattaaatatagttttggaacttg aatattagtatggttaataagtgctgatatgctgatt ttgtttatatttttcttatgagtaaatatcctatatc accagacattatagtctatgtacaaatatgattctta aacctgatagcacattcattagagttggaattgcctt ttttttttttttttttacagttgcacctgcaacatat gaaagttcccaggctaggggttgaatccaagctgcag ctgccaccctacattacagccgtagtaacagcagatc cgagctgcatctgcaacctatgctgcagctcagggca atgccagatccactgagtgaagccagggatggaactt gcatcctcatagagacaacgtcgtgtccttaacccac tgagccagaacaggaactccagaatttcctttcaata gaagaagcaccaagtttaggatcagaaagcctgaatt tgaataccaatttactatttgttagtcatatatttct gagtgtgtttcctcatttattaaaagcagactaaaag atgagagggtcttttgttgagaatcaaatacaataac atgtgaaagtgtgtaacactatgattgaaatatacct acacagccatttatttgtttattgttcatgttttgcc acccacacagtagtatataatccttttatgtaataaa tgctaataatgaaagttggcaacttatgtaagtactc aaaatgctggaggtcatgggatactgactgggatact acagaggtaatgtcatttcctctgcgctaaacttatt gtctgtagttagggactgactctctttaggacaagga gttcattctgtataccatgtgtggctatcacccttcg aagttgaaaaactgccccagggtgggcacccatccgt tctcttagatatatggccgagacctttctctcactgg gagggaaccacactgaggaatgagaaaaaaaaaagga aaatcaagatgaaaccagaaacctctttggcataact tctccactctgtactttttgttagaactacccttgca caaagcagcatcagtgtggaagacagaatttgcacac ctggtttgatatacatgccgtggtatatgggatgttc taacaataaagaggactctcccaggaaatctcctcac tgttatagtcagccttgaggaaagagctcttcttttg gactctggggagagtctagtttttcagttccttgctt ctcggtcaacgtgttggtgtaaggatcacactctctc ttatactagataattctattttttcacctttcaacct gtctatccttctgaccctagTTACCCAACACTGAAGA AGCTTGCTGAGAGAAGACCAGATGTTCCCATTTATGT TGGCAACACGGAAAGACCTGTATTTTGGAATCTGAAT CAGAGTGGCGTCCAGTTGACTAATATCAATGTAGTGC CATTTGGAATATGGCAGCAGgtctgtgttctttccac atgtttgggttatcctttctgggataaatttgaggcg agatagaaactttaagactaaagaaacaatggcctac tttttttgtacatggtcctgtgtaaatctctatttga gctgaaataagatggtcttcctctccaattatccatg gtatgactctgatggataacaaatccagttctgaaaa aaggggatttctttccagaagagaggacagtttcttc aaatattgaattaaaagcaaaatagatgtaaaccgtt gttggttttattgttgaattccagGTAGACAAAAATC TTCGATTCATGATCTTGATGGATGGCGTTCATCCTGA GATGGACACTTGCATTATTGTGGAATACAAAGgtatt ttcttgccctcatcagcatgaaattgctcttggtaga aaggataataatagttatccaaaacatcatcctatgt tcatctgtttcttccctcttcattttccatagagtac agtatattctatctctgtcttaggaaaatggactgtc attcatataatcttacagagaatcaattagtaatgta ctctatgccgtgacaggtgcgaaggttttttttgaag gcaacagataaaaatatcctatatttcacctattgta atttccttaaaactgacattattgaataaatgtttta ctttcatcttgaatattattatgttatggaatcatac actttaccccaataatcatcgaaaagaatttccaaaa ggttgagagagttgtgttgatctgattactttcctct gcatcctttgagcttaacctttgaatatagtttgcta aggaaagtagtctgtttatgatcctggagtggaatca ggctaagtgtcctcattcagaacccactgaatcagac agaatgaatttatttccttgaaagttcaaaatgtgtc actcaagagtataaattttcaaatcttactctctctt ttccttggatgtgagcaattcttcgataattgaatga ggcagattatatagacttacatggaagactgttggcc tgagaattcaaactatggtgttcaagacttcacngng agtccgatgccatttgtttcccacagGTCATAAAATA CTCAATACAGTGGATTGCACCAGACCCAATGGAGGAA GGCTGCCTATGAAGGTTGCATTAATGATGAGTGATTT TGCTGGAGGAGCTTCAGGCTTTCCAATGACTTTCAGT GGTGGAAAATTTACTGgtaattctttatatcaaaatg atgccaaggagttggcatggcactttgctaaatgctg tgtgaatcaatacaaagataattaggacatggttctt cctcacaagaggtgtgcaatcttattgggaaatcata cttgcaagtcacaaatatagactaaagtttccagctg agaatatgctgatggagcatgaaacactaaggagaca gggagaatctcaggaaaaatcaagaataatttggatc aaatggattcctgacatagaacatagagctgatcaga aagagtctgacattggtaatccaggcttaagtgctct ttgtatgtggttcagaacagagtgtgggcagcctgag ggggatacatacccttgacctcgtggaaagctcatac gggggagggatgaggctaaggaagcccctctaaagtg tgggattacgagaggttgggggggtggtagggaaaat agtggtcaaagagtataaacttccagttacaagatga ataaattctaggggtataataacagcatggcactata gatagcatattgtactatatactggaagtgctgagag tagatcttacatgttctaaccacacacacacacacac acacacacacaccacacacacacaccacacacacaca cgtgcacacaaacagaaatggtaattatgtgaggtga tggcggtgttaactaactttattgtggtcatcattta gccatacatgcatgtcatgaaatcaccatgttgtaca ccttaaagttatgtaatactagatgtcagttatatct caaagctagaaaaaatgtggggaccaaggcagaagct cttctgctctgtgtctaagggtggttctggggctggg atggggaggatggttaagtggtatatttttttcatac ctttgctcagtactatcattgtaagtgttcaatatat gtctgcttaataaattaatgtttttagtaagtaatct ctgtttagtaatgtgtcagaaatgccctacttgcaat aggaagaaaacctgtccagtcccttccttttttctgt aagtctgatttcattgcctcccagaatgcatcaccat gtgagagatagagggaaggtgctgtccttatggggtt aacagtgtgactagggaggcaaaatatacctactaaa gggtggtagcataattcagttcttatgtgagtatgtg tatgtgtgtgagtatgtgcacatgcacatacatttta aaaggtctgtaatatactaacatgttcatagtggtta cacctagcttataggtaacattttttcccctgtatcc ttgtttgtgtttatcaaattttcataacagtaatggt agaaggagtacctgacatggtaccatacatgctnggn cctgcctaatttctcnatttcctttattgcccatacc cccattgcttgacaagcataagtccatactggcttgt tttcgttcctcagactcagtacaccatgtagctccat gccctgggtctttgtatgtgctatttctactgcttag agtgctattgcccctgaccaccacgtggtcagcaact tctcttctgcgtctgtgtctatggtctatgattccag atgtcatcttcactaactacccttctaatatgccctt ccatcccacccgtcctcatccttaccccagccactct ctatttggtggctctgttttattttcttcctagctca tcactctttgaaatgaacttatttacttattcaatat ttgcttctttcactagaatgaatgctccatgagagca gggacctgctttatcttgctcgccactgtattcacag tgcctagaactacgtctggcacatagtaggtgctcaa taaatatcgatcaaatgaaagaatgagcaaacgaaca aatgaacaacacgtgaggtaggcatcatgattccatc aacagaggagaaaaccagacttaaagnaatgaagtgg nggagctgcatttgatcttgactgactccaacatcca tgctcttgaccactgtgcatctccagagtgtaatgaa catactttacttttatattccaccaaaataacaaagc catgcccatgttagtagagagttaatcgacagtgccc ttaaaatatgcatgcacccagggtacaactatgcatg ctgccctgtgttttcagttggatccaaatgaattgcc gtaaacaaagaggggattcaatgtctttgactagttt gggatattttcctagtaaccaactttgcaaaataaag ccactaatgacaaggagctttgttctacttctgcatc actcaactgtcaatttttatctcttgcaagacttcta atctactagaacttttgtttttctgtgatttctgaac agagaagactaatccaaaccctgtcattccagAGGAA TGGAAAGCCCAATTCATTAAAACAGAAAGGAAGAAAC TCCTGAACTACAAGGCTCGGCTGGTGAAGGACCTACA ACCCAGAATTTACTGCCCCTTTCCTGGGTATTTCGTG GAATCCCACCCAGCAGACAAgtatggctggatatttt atataacgtgtttacgcataagttaatatatgctgaa tgagtgatttagctgtgaaacaacatgaaatgagaaa gaatgattagtaggggtctggagcttattttaacaag cagcctgaaaacagagagtatgaataaaaaaaattaa atacaagagctattaccaattatgtataatagtcttg tacatctaacttcaattccaatcactatatgcttata ctaaaaaacgaagtatagagtcaaccttctttgacta acagctcttccctagtcagggacattagctcaagtat agtctttatttttcctggggtaagaaaagaaggattg ggaagtaggaatgcaaagaaataaaaaataattctgt cattgttcaaataagaatgtcatctgaaaataaactg ccttacatgggaatgctcttatttgtcagGTATATTA AGGAAACAAACATCAAAAATGACCCAAATGAACTCAA CAATCTTATCAAGAAGAATTCTGAGGTGGTAACCTGG ACCCCAAGACCTGGAGCCACTCTTGATCTGGGTAGGA TGCTAAAGGACCCAACAGACAGgtttgacttgaatat ttacagggaacaaaaatgatttctgaattttttcatg tttatgagaaaataaagggcatacctatggcctcttg gcaggtccctgtttgtaggaatattaagtttttcttg actagcatcctgagcttgtcatgcattaagatctaca caccaccctttaaagtgggagtcttactgtataaaat aaactattaaataagtatctttcaactctggggtggg gggggagactgagttttttcacagtcctatataataa ttttcttatcctataaaataattaggagttcccgtag tggctcagcaatagcaaacccgactagtatcgatgag gatgcgggttcgattcctggcccccctcagtgggtta aggatctggcattgccgtgagctgtggtgtaggtggc agacacggctcagatcccacgttactgtggctgtggc ataggccagcagctccagctctgattagacccttagc ctgggaacttccatatgctgtgggtgtggccttgaaa aaaaataaataaataagataattactcaaatgttttc cttgtctcagaaccttacttcaggataaagagtgaga aagttttttttatgaagggccattattacagctcaaa aataagttgtcttcagcaagtagaaagcaataagcct gagagttagtgttcctatcagtgtaaatattacctcc tcgccaatccccagacagtccatttgaacaattaacg gtgccctgggagtacagttcagaaacattaatgtgga tgttccagacctgtatttttataagtacttgtcttga gccggatggaaccatcattcctcaccattatttagaa gtggactgtgactctgttggagatcagggcacacggt taccaaaagcacacccttctcctggccttacctttgc aaagctggggtctgggacacagtcagctgattatacc cttttactaacttcccacagctcaaatctggtcaatt ctccttcacaaatctcttaaaaatccatcactcacct ccagcctcttctgctgtggccttgattcagcctctca caatttttttttaaccagaattctggcagtggcccct gacttgcctctgtgctcccagccccgctgtcctctga tccatcctccatgccagcctttttcaatctgctggtc acgattcattgatgggttaggaaatcaatggcatcac aactagcatttagaaaaaggaaataggcgttcccgcc gtggcacagcagaaataaatccgactaggaaccataa ggttgcgggttcaacccctggccttgttcagtgggtt aaggatccggcattgccgtgggctgttttgtaagtca cagacatggctctgatccggcattgctgtggctctgg cgtaggcctgcagcatcagctccaattagacccctat cctgggagcctccatatgctgcaagtgcagccctaaa aaaaataaaaaaataaaaaaaaataaataaaagaagt agacaaattgtatagaacaaccctgagtatgttgcct gagcacatataacaagggtaagtattatttcaggaaa ctctggtttcacagatactcttggcatatggacccct agagtcctgatgtaaaatatattcttcctgggatctt aggcaagaagtttgaaagctccaactctgcactgctg ccaaagaaatgatttttaagtgcaaaactcttcccgt tcccttccctgtataaaattccataggatctctccag tgcctctaggataaaggcagttttcattctctagttc aaggtgagagaagattttaattatttcacgttttagt ggggaattcaagagtctggcacctgacatttgctgaa ctctctccattatccctctctagttccccagacgcat cctatggtagaaattcgcaaactagagtgagcgtcag agtaacccaaggaaactgggtaaatgcagctccctgg gctctaccccctgagattctgattcagtagatctgaa gcagagccctggaatatgcatatgcatcattgtgtca caccaagcattctgggtaatgagagttgatgttaggt tctcagtagtaagacaagtatagagattccgggggac tgagtgctcagctctgccttggggaggagggagaggg ctaaagagaacaggagatggggacagggaatgctcaa cctccaatcttaggcatttgagctatgtcttaggggt caggaggaggttaccaatatagtgattaagagattga ggttccagtcagagggatatgctggagaaggggggtg aaaataatgtcataggtttggtgagtgcagatacttt gagttttttaatatttttattgaaatatagttgattt acaatgctcttagtgagtacaattactttgaataagt gcatagatgtatgccattcttccagaaatgatttatt gagctcctttgggcatcatgctaagtacaggggaaac agctgtgaagaggtccttcccttatgaagtcattcat ccccttcagtaaatgaaggtaaaggaaaaggatgaga cagggacgccgtgttggaccagggtcagaaaggcctt ataagaccttgcctggagggcaaggaacttgcctgtg agtaaggagagcttgagaaagcgataaagcaaagaag gaacattactgcattgtgttttagaaaaaccatgtcc tggggaagaactcctagagtcaggggggccagttggg agactgtgcttttttccaggaggagataagtgaggct gctggctgagatggagcaaggatttagagaagcagat atgagattcatttagaagttagacattttaggatctg acacataatttatcaccaaaaccagtgcatctctggc tttgggccaccagttttggagaagtggaatgtaggga cctaccattacctgccaatctttactacacagatgcc tatttccctcctcatatttcctttctccagatcacgt cctattctattgccaggactcaagattccaccttgca tgcagtgatccatcttcacactggatggacagctcta gggatgtcagagcacactcccatactgctgactgggt ctcctgtcagcccatctgtctatcagctgtggtatta ttagtataataagagggctgtatatgagagacacaaa attctaggtgtagctcaaagataggctagagttattc ctatgtacaacaaatatttatgggaccccttctgtgt actgtcatggttgctgctttcatcatacttgtagtct aatggaggtgggggcagggcaggaataagcggatgtc cacaaaatcagtaagaccacttatattcaacattttc ataatttagttatttgagcccaaagggtccacatccg tggtattccaacttttttttccccggacatggatctt tatctttttttttttttcttttttgcggccagacctg cggcatatggaagttcccaggccaggggttgaatggg agttgcagctgcctggtctacaccacagccacagcaa ggtgggatctgagctgcatctgtgacatacaccgcag ctgaggtaacaccagattctgaacccactgaatgagg ccagggatggaacccgtctccttatgaacactatgtc atgttcttcaccctctgagccacaacgggaactccag acttcgtctttaaatgtattctgacttggagagctat cacactaagcaattaacaggagctgacctggtttagg ctggggtggggccctactcctcaatgttccctgaggc acatctgtgggacccctgggcatcatctatctgagca gccttagagctgctcatccagttgactgttgatgtag aagtgcaaacttctgccttccttatgcttlctttttt cattgttctctcccctttgtgtctttaagCAAGGGCA TCGTAGAGCCTCCAGAAGGGACTAAGATTTACAAGGA TTCCTGGGATTTTGGCCCATATTTGAATATCTTGAAT GCTGCTATAGGAGATGAAATATTTCGTCACTCATCCT GGATAAAAGAATACTTCACTTGGGCTGGATTTAAGGA TTATAACCTGGTGGTCAGGgtatgctatgaagttatt atttgtttttgttttcttgtattacagagctatatga aaacctcttagtattccagttggtttctcaataagca ttcattgagccttactgactgtcagacggagggcgta ttggactatgtgctgaaacaatcctttgttgaaaatg tagggaatgttgaaaatgtagggaatgaaatgtagat ccagctctgtttctcttttggaggattctttttcctc catcaccgtgtcttggttcttgtttgttttgggtttt tgtgggtgttgtattgtgttgtgttggttatggcagt gacagctatttaaactgtgaaacgggggagttcccgt cgtggcgcagtggttaacgaatccgactgggaaccat gaggttgcgggttcggtccctgcccttgctcagtggg ttaacgatccggcgttgccgtgagctgtggtgtaggt tgcagacacggctcggatcccgcgttgctgtggctct agcgtaggccagcggctacagctccgattggacccct agcctgggaacctccatatgccgcaggagcggcccaa agaaatagcaaaaagacaaaataaataaataaataaa taagtaagtaaaataaactgtgaaacggggagttccc ttcatggctcagcagttaacaaacccagctaggatcc atgaggatgtaggttcgatccctggccttgctcagtg ggttaagaatccagcgttgctgtgagctgtgatgtag gtcgcagatgcagcccagatcctgcattgctgtggct gtggcgtaggctggcagctgaagctccgattcaaccc ctagcctgggaacatccatatgctgcaggtgtggcct taagaggcaaaaaaataaaaaaataaaaaataaataa attgtgggacagacaggtggctccactgcagagctgg tgtcctgtagcagcctggaagcaggtaaggtaaggac tgcagctgggtaaggactgaattgcaccaactgggaa gtaagcctagatctagaacttaagttagccctgacat agacacacagagctcaccagctaagtggttcagctta taagctggtcactgaaactgaggatgtccacaaaagc aaaataagtagcaacaggcagcgggatgcaagagaaa gaggaggcctaaaatggtctgggaatccctgccatac ctatattttatcctacttatatttagtgcctgaatgt gtgcctggagagcaaagtttagggaaagcatcgggaa atgcacagtattcatacccttaggaacaaagatcagt tacctccagggtaaagactatttccaagtttaaattt caacccctgaacattagtactgggtaccaggcaacac ttgccatcctcaaaatcaatgaatcctaaaattcaac ctgggggtcagtgacagtctgtgacaaagtttttgct ggtcagtaacgaaataagtatgagcaccatctgagta tggtcaccaagatgtcaactctctttcctttggacga attgtcattattccaagattaggtcctttctattttt gaggtgtgaaaacatctttcctttcataaaataaaag gatagtaggtggaagaattttttttgttttttggtct ttttgctatttctttgggccgcttctgcagcatatgg aggttcccaggccaggggtcgaatcggagctttagcc accggcccacgccagagccacagcaacacgggatcca agccgcatctgcagcctacaccacagctcacggcaat gccggatcgttaacccactgagcaagggcagggaccg aacccgcaacctcatggttcctagtcggattcgttaa ccactgcgccacgacgggaactcctaatgatactctt ttatatttagctactatgtgatgatgagaaacagtcc acattttattattttttagccaatttgatatctcatt actaagataatgataattttctctataaattttattt aagttagtgttatgaagtggttttgctagtgtagaag gctaggatttgaattcagttcaagaaagaagagaggg agggagggagagggatgggtagagggatggggcagtg ggagagagcaaagaggagagacagtttttgtattaat tctgcttcattgctatcatttaagggcacttgggtct tgcacattctagaattttctaaggaccttgaccgcca gattgatatgcttcttccctttaccatgttgtcattt gaacagATGATTGAGACAGATGAGGACTTCAGCCCTT TGCCTGGAGGATATGACTATTTGGTTGACTTTCTGGA TTTATCCTTTCCAAAAGAAAGACCAAGCCGGGAACAT CCATATGAGGAAgtaagcaggaataccagtggaagtg cccctttcttccttccttcctaaataaacttttttat tttggaacaactttagagttacagaaaagttgcaaag atattatagacagtagtgtttatatatatatataaat ttttttttgctttttatgaccacacctgtggcatatg gaggttcccagtctaggggttgaattggagctacagc tgccagtctgtgccataaccacagcaatgcaggatct gggccacgtctgtgacctacaccaaagctcacagctg gattcttaacccactgagcaaggccagggattgaacc tgcatcctcgtggttcctagttggattcgtttccgct ttgccgcaatgggaactccaaattattgttaatatct tactttactggggtacatttgttacaaccaatactct gatactgaaacattactgttaactccgtacttgcttc tttttgagtcatttgcaaagactggcttcttgacctg cttccttccaaacagctggcctgcctatgctgttctc agacctgcaagcactgatctctgccccccttgccttc tctccagtggtgtctccttccccaaacaaacccagtg tggctctggaaagggagttaagtcaacataaaccaac acatattttgttgagctccaattttgagcaaatccct cacctacggcagacaggcatgatgttaagaactaggg ctttggacacaaggtcaagaccaagaagggttcctca cccctactgattcagataaccaataatgaggctttga atccctgtccaaaggttgttttttttcccttctattg agcttcttgccaccttatcagttttttttatgacagt caaatgacatgatatatgtgagcatacatggtaattt ttaattctatataaatgaatcactaaataaataggag gatatatagtccacctttaagcgtattacacgtgtca catgaatgtggcgacttaattgtagaggtttaaatgt agcttcctataatagatgtgttcctaaactacatttt aatcattggacttgtatttttatgttagcacttgctg ttgaagaaaagcctatgccaaaagttcagtgaaacca ataatccactgccagctttctgagttaaaaaaaatcc ctgggttttcacacacaggaacaccctgtgtgaaaca ctcatttagagcaaaatgcatctgataaggagttcct gttgtgcctcaactggttaaggacctgacattctcca tgagaatgtgagtttgatccccggccccactcgatgg gttaaggatctggtgttgccacaaactgcagctccga ttcatctcctagcctagaaacttccacagcccagaat atgccacagaattcggctgtttaaaaaaaaaaagaaa aaaaaaagaatcataaatgtgttggtttgttcaccaa atacatgataacttgctcttgccaagctcagcttcat aaatattaagtcatttaatacagcagccaccttatga acagatattactatacttcccatttacagataaggaa aatgccatatttaaccaagagattaaataactttccc gaggtcttatagcaagtaaatcatggtgcaggggttt gaccacacgcagtctatctccagagtctgtgtattta gccactgttttactttcaaatttaaatttataaaact tctaaattatctgttaaccataatctttggaattttt aaaaccacgagttcctataaaatgtttcattgaaagt aagtcacttttccatagcttttgataatacatctgta ggataaagtaagccacagctctcttgcagacttggta caccctggggcaaagcatcatgcctgtcacgtacatg gtggtccttactttgactctcagtgcttttattgccc aggaattttgtgagatttctagttgttgaggtttgtt taaagaggttatgccggtacttggaagagctcttttc ttgctacctggagccttctcatatttcctttttgagg agggacatgaattgcctttcaaactcataaatatatt ttctagtacacaagtctccatcttccttagacgcatg gctcctggagttctccatcctcctgctccactttggg tgggctcctctctgggtctgccaccaatctgccaccc agagacatccttgacccacttccagaccccaccatgg cttcactttcttcgctttcctcctttgtggaaccttc tgcttaagaatctgaggaagaaaatttgcacgtgagc taaactggaggtactttcctgcctggtcttgcacgat agcttggctgagcccatgatgctgggtggctgttact ttccatggacacccgaaggcgttgctcctttggcttc tagttgcatgcagtgttgcttatcccaggctgatctt tcttccactgtaggtgacttttaagaattaagggatt aatctatatctacaacaacaacaacaaagaccttttc aagctgaggtagggctttctgtatatgtttggagtgg ttatccagcagactttacttgaaggcaggggtcatat cctcaagtgctcataaacggaccacagaaagatctca taattgggtggagctgggtggggaccgtgtcatgtgg ccaggaaatgccagatgggaagggagtggcccttact gagctccagctgaactctgaattttctagaaaactca gaaatctggatttttcatgtgtaatacccagatttat agatgtggaaagctaattctttttttttttaagggac tataggcaatgaactaagatctaggttgtatttggac aaggggtcatcagtttaagctgtgtagttgagcgctc agctattgggctgagggacccctaaatactgagacgg ggaggtccttgctctggggcatcacaagtacactccc tggtctcattcaaacacttttcctacaaaattgatcc catttcttcagtgcactgtctgaatgcatttggccca gagccgtgctgaggcatagggaaggggtccacggttt catggcatcgttttgtgctgtgtgtccctgctgtcgt ccaggatacctacctctcctcctcctgcatctgaatg tccccccacagactctctgggattctacagcctctgg cctgttcctcagacacctcttacctgccagctttcca gattcacattagttagtccaaatctactgccgtcagt gactcacttcatttcttcttctccgaggcagttcagc ccggtacagttgttttgtcaacacttcagttgagtct ggaagatgtgcatgggttatgcacgagagcggtccat cattttgagctagaagtcctttctcagcccagagaca agtcctcatctcctttacttcctgactcttcttcctc tgcatccttccaagatatctctttctccagccaccac ctaaatctcttcttttcccggggttccgtgctcaacc cactcttcttcttaaatctgtggctgggtgaacgcat ctgctggcaccacttctctgctaagactccaaaaatc cataggtcctgcccggcctttgcccacctctctccaa cactgtccagctttagatgtagagctaatccccccag agatatcattccctggatgtctaagtcctttggtatc tcactttcagcgtgttcaaaatcctcttacaactgtt ctttctccttttccatcttgattattggcaacatgcc agcctttcccctacccccagcagtgagccaagctaga aacaagggcttaatcttcaatctttccttctccatcc ctaaacctaatgagtctccaagcccttcccagtttac accctaaatgttgctcaaaacatcccctagttcttcc acgtgctctcctctatattgaaaggtcaagaaaggcc atcttccctccactgtgaggaaatagatcttgatact gcccctgagctgggcagtcctcgacctgacaaactgt gcagtgtttctaaatctctactggcaaaatgagagtg cctttgacctgtgttgcgatctcagatcacagtggat gtaattgttttataggaatggtgaacgaaaaagaagt aaatccctaatgccaaactcctgatcattctatgtca tttaatagcctgtcatttatgataaagtttcctctac tggcattagcacaatacttctcaggaaaaaaaaatat gatgccagatactgaaaagctcctgggtaaacatgaa catgggtaccgataaaatggtgaagccagtccaatct tagagtgacttcccttcatgctacttcatgctctttt ttttttttttttttaagaaaaaccccttttttttttc tcacaccagtcacagaggagaccgaggcttagcaagg ttaaggtcacatgattagtaagtgctgggctgaaact caaaaccatctctgcttgtctcctaaccctgtgcacc tctgactattcaacagATCCTGTGTCAGGAGTTGGGA TTCTTTGAAGgtaagggccttgaccaccgaattaagg taatcttgctctgtggcaggccttgttttcagtattt taagtacactggctcaggtaatcctcacaacagcccc aggaggaatgttctattacctccactgtatagatgag gaacttgaggcacagaatggttgccaaggtcacacag ctatattgggggttcatacccagccatccaactctgt ctgtactctctgccactctgcacccccagctcctgat ccacttcctgtttccatccctcgatttctgctgcact caggggcccctctccccctcggcctgtgagatctgct tcagtaggcttttctccctgactcctccatccctgtc cttacaggcagctgcttctctccgggacacgaggggt ccatacggacactctctactggctgggttgcgcctaa ctcgtgattcctcctctgtttcagATTCGGAGCCGGG TTGATGTCATCAGACACGTGGTAAAGAATGGTCTGCT CTGGGATGACTTGTACATAGGATTCCAAACCCGGCTT CAGCGGGATCCTGATATATACCATCATCTgtaagtcc gaaaatgcctgtcgtgtgtgccttaggctgctgcgga ggaggccagggctatataagcagagtcagtgactgac tgtgccctgcagtgttgatggccatggagattccacc gttagagcttttttctttgttaaccttgaaggcaaat ctggttaggaagataactttcaaagagtcaccatctg gacattcatgcccatgtgcttcaatcctgtatacaag cagtttagagtacagggaagggaaggacattatgaaa gggagagggtgtgtttggatccagcagctccatcctc agaatttatctgaagacactgcaaaattactaagaat cactatgacaagaatgaggatggggtgatatggcaaa gttgtgatcctggaagaccttcatctcccatgttgcc caactctgaacatgaatttggtgaactagttggttaa ggggatgatcctccaagtttctccctggttgagctcc aaaaaccatgtaagtttctcatagcaaaaccgtatag gtccttagggctttagttggaatatttgtgctgaaat gctggaaagccccatttgccatttttgtatttgcaaa ataatcatcaagaggggagaatgcattctttcatgac cactgaccctctgaaaaggtcaggaatttagtctgaa gtaggcaagcctcctaccccgcttctgccatgagctt gcacgcacaggcctgtcttgacatttcttctttatag atttctttttgaatatcttgaaattgctttaaaaata tttaaagaatgtagaattatataaaataaaaaggaaa taaccccacacctcccacaaaaccctgtttcctgcct ttctccacccactctccagggtaacacttggtaacag catagttgtatcaccccaggcctatttttgagcatat cagcatttcaagaaatgtattttttctcaataaaaca tcccttatagttgaggaggggaggttatcattcctgg gttttgttttttttttttttttaatgtaatcctggta catcggtaatttgcattttttattcattaatatcttt ggtatttctagtgttgggacacacaggtcaacctcag tttttgggtttttttttttgtctttttgtctttctag ggccacacctgcagcatatggacgttcccaagctagg agtctaatcagagctgtagccaccagcctacgtcata gccatagcaacgtcagatccaagccgtgtctgtgacc tacaagcacagctcatggcaacaccggatccttaacc actgaacgaggccaggggatcgaacacacatcctcat ggatcctagtcatgttcattaaccactgagtcatgat gggaactccaacttcaactattttaatgtctgtaaaa cattccatttggaaaccatttcatttgtaaagcaaaa tgaaaacattttgttcattttcaacagagttcgtagc tgacttctgttctggaaaaaaggaaatggagcaaatt tgagtgagaaagattcaaagataacttttcttttaaa aaaaattatatcttggaaacttctgggctattgattc tgaagactatttttctatatactgttttgatagcaaa gttcataaatgtgaaaggatcctgcgatgaatcttgg gaagcagtcatagcccaatatatctttgttgctttta aaatgagatttagtttactaaatatttttctgatcat aaaaataacacagatctaccgcagaaaatttggaaaa aaaaaaacttttaaattcaaaaaacagttaaaccaca aatgatcccaccatccagagagcaatttgtactttgg tgtctagttcatctttctttttctgtttacaagcaca tataccacaagcattttttcaaaaaatgaaaatggga taatactatacatacgtctgtacacctgcatagttac tgaacagtctttgatctaccctgtaagtttctaactt ttcattatttgaaatgatgttttggcaaagaaatatg taggtgtgtctcgcacactttcataatgatttcttag gataaatttcttaggataaattcataatgatttctta taataatccatactctgccaactgatcttcagggaag ccaactcgccttctcagaaataacatataacccattt acttgccctctcaccaatactaggtcctaatgttttt gtgtacagattctatatttttacatacaagaattcct taaagcaaggcatgtcacagaaaaatagaaggaagac acaattgtcatgtttaaggactgcattctgtaccaaa aatgctaagttaaatgaacatctgaaacagtacagaa acgctatctttcagggaaagctgagtaccaggtactg aacagattttggcaaatacagcaggcatggatgtttc caaaacatgtttttctactttatctcttacagGTTTT GGAATCATTTTCAAATAAAACTCCCCCTCACACCACC TGACTGGAAGTCCTTCCTGATGTGCTCTGGGTAGAGA GGACCTGAGCTGTCCCAGgtaaagcatcctgcaggtc tgggagacactcttattctccagcccatcacactgtg tttggcatcagaattaagcaggcactatgcctatcag aaaacctgacttttgggggaatgaaagaagctaacat tacaagaatgtctgtgtttaaaaataagtcaataagg gagttcccatcgtggctcagtggtaacgaaccctact agtatccattgaggacacaggttcaatatctggcctc actcagtcggctaaggatccagtgatgccgtgagctg cagtgtaggccacagacgtggctcagatctggtgctg ctgtggctatggtgtaggccggccccctgtaactcca attcgacccctaggctgggaacctaaaaagaccccaa aaaagtcgctttaatgaatagtgaatacatccagccc aaagtccacagactctttggtctggttgtggcaaaca tacagccagacaaacaagacaaaaattatcctaggtg gtcagtgggggttcagagctgaatcctgaacactgga aggaaaacagcaaccaaatccaaatactgtatggttt tgcttatatgtagaatctaaattcaaagcaaatgagc aaaccaattgaaacagttatggaagacaagcaggtgg ttgtcaggggggagataaggggaggcaggaaagacct gggcgagggagattaagaggtaccaactttcagttgc aaaacaaatgagtcaccagtatgaaatgtgcaatgtg ggaaatacaggccataactttataatctctttttttt ttttgtcttttttgccttttctaaggctgctcccgtg gcatatggaggttcccaggctaggagtccaaacagag ctgtagctgccagcctacaccagagccacagcaacac gggaaccttaacccgctgagcaaggccagggatcgaa cccgagtcctcacagatgccagtagggttcattacca ctgagccacgacaggaattccagggtctgttgtgttc ttaaaacacttccaggagagtgagtggtatgtcataa gtaaacaataaatgttaaccacaacaagcttatgaaa taaacaggaaagccatatgacctacaatcagtcattg ggagaatccacaaaaggttgagcagaggatcaattcc agctcacactccagttttagattctcccctgccttaa agcatcacagactacataatctgagctgaagaataaa aattaaaactcaccccagtgcaaaacagaaatgaaaa agtattaaaacgaggttcatactgttgttcattagca atatcttttattcacagGGGTGCCCAACAACATGAAA AAATCAAGAATTTATTGCTGCTACGTCAAAGCTTATA CCAGAGATTATGCCTTATAGACATTAGCAATGGATAA TTATATGTTGCACTTGTGAAATGTGCACATATCCTGT TTATGAATCACCACATAGCCAGATTATCAATATTTTA CTTATTTCGTAAAAAATCCACAATTTTCCATAACAGA ATCAACGTGTGCAATAGGAACAAGATTGCTATGGAAA ACGAGGGTAACAGGAGGAGATATTAATCCAAGCATAG AAGAAATAGACAAATGAGGGGCCATAAGGGGAATATA GGG

TABLE 11 Contiguous 5′ Genomic Sequence of CMP-Neu5Ac Hydroxylase gene ctgccagcctaagccacagccacagcaacgctgggtc Seq ID No. 47 tgagccatgtctgcagcctatgccagagctccccgca gcgccggatgcttaacccactgagcaaggccagggat tgaaccctcgtcctcatggatagcagttgagttgttt ccacggaactcttaggggaactcctgattatttttta tttaaatttatatttctctgactttttcgtgtgctca tcagccactgactgtgtatctccattagtcatggttt gttaactctgtcattcaaaccctcttcatccttgcta cgcagataacatcattataataaaatcgtgcctgaag accagtgacgcccccaagctaagttactgcttcccct ggggggaaaaagaagcaccgcgcgggcgctgacacga agtccgggcagaggaagacggggcagaggaagacggg ggagcagtgggagcagcgggcagggcgcgggaagcac tggggatgttccgcgttggcaggagggtgttgggcga gctcccggtgatgcaggggggaggagccttttccgaa gtagcgggacaagagccacgggaaggaactgttctga gttcccagtCCCGACGTCCTGGCAGCGCCCAGGCACT GTTATTGGTGCCTCCTGTGTCCACGCGCTTCCCGGCC AGGCAGCCCTGGCGGATCCTATTTTCTGTTCCCCCGA TTCTGGTACCTCTCCCTCCCGCCCTCGGTGCGCAGCC GTCCTCCTGCAGTGCCTGCTCCTCCAGGGGCGAAACC GATCAGGGATCAGGCCACCCGCCTCCTGAACATCCCT CCTTAGTTCCCACAGgtgagaaggcttcgccgctgct gccgctggcgccggcagcgccctccacgcacttcgta gtgggcgcgcgccctcctgcattgtttctaaaagatt tttttttatccgcttatgctatcagttactgaggaag tatttacaaatctactattattttgaatttgcctttt tctccttatagtttatcagtatctcttgagactgtta ttggtgcctgcaaatttaaaatgattggggttttatg aggaagtgaaccttttatctttatgaaacgcctaact gaggcaatgttaattgcttaaaatactttctttatta tcagtgtggccatgccagtgtcctcttggttagaatt tgcctgat

TABLE 12 Contiguous 3′ Genomic Sequence of the Porcine CMP-Neu5Ac Hydroxylase Gene ctgccaaagctgggagatgggggaaagtagagtgggt Seq ID No. 48 tattgaaactgaatatagagttcagcatctaaaagcg aggtagtagaggaggaagctgtgtcaacggaaatact gagctgggttcacatcctctttctccacacagTCTAA TGCCTTGTGGAAGCAAATGAGCCACAGAAGCTGAAGG AAAAACCACCATTCTTTCTTAATACCTGGAGAGAGGC AACGACAGACTATGAGCAGgcaagtgagagggggctt tagctgtcaGggaaggcggagataaacccttgatggg taggatggccattgaaaggaggggagaaatttgcccc agcaggtagccaccaagcttggggacttggagggagg gctttcaaacgtattttcataaaaaagacctgtggag ctgtcaatgctcagggattctctcttaaaatctaaca gtattaatctgctaaaacatttgccttttcatagCAT CGAACAAACGACGGAGATCCTGTTGTGCCTCTCACCT GCCGAAGCTGCCAATCTCAAGGAAGGAATCAATTTTG TTCGAAATAAGAGCACTGGCAAGGATTACATCTTATT TAAGAATAAGAGCCGCCTGAAGGCATGTAAGAACATG TGCAAGCACCAAGGAGGCCTCTTCATTAAAGACATTG AGGATCTAAATGGAAGgtactgagaatcctttgcttt ctccctggcgatcctttctcccaattaggtttggcag gaaatgtgctcattgagaaattttaaatgatccaatc aacatgctatttcccccagcacatgcctaactttttc ttaagctcctttacggcagctctctgattttgattta tgaccttgacttaatttcccatcctctctgaagaact attgtttaaaatgtattcctagttgataaacagtgaa acttctaaggcacatgtgtgtgtgtgtgtgtgtgtgt gtgtgtttaccagcttttatattcaaagactcaagcc tcttttggatttcctttcctgctctctcagaagtgtg tgtgtgaggtgagtgcttgtccaaacactgccctaga acagagagactttccctgatgaaaacccgaaaaatgg cagagctctagctgcacctggcctcaacagcggctct tctgatcatttcttggaagaacgagtgctggtacccc ttttccccagccccttgattaaacctgcatatcgctt gcctccccatctcaggagcaattctaggagggagggt gggctttcttttcaggattgacaaagctacccagctt gcaaaccagggggatctggggggggggtttgcacctg atgctcccccactgataatgaatgagggattgacccc atcttttcaagctttgcttcagcctaacttgactctc gtagtgtttcagccgtttccatattaggctcttccac cgtgtcgtgtcgtcaatcttatttctcaggtcatctg tgggcagtttagtgcgaatggactcagaggtaactgg tagctgtccaagagctccctgctctaactgtatagaa gatcaccacccaagtctggaatcttcttacactggcc cacagacttgcatcactgcatacttagcttcagggcc cagctcccaggttaagtgctgtcatacctgtagcttg cttggctctgcagatagggttgctagattaggcaaat agagggtgcccagtcaaatttgcatttcagataaaca acgaatatatttttagttagatatgtttcaggcactg catgggacatacttttggtaggcagcctactctggaa gaacctcttggttgtttgctgacagactgcttttgag tcccttgcatcttctgggtggtttcaagttagggaga cctcagccataggttgttctgtcaccaagaagcttct gcaagcacgtgcaggccttgaggtcttccgacttgtg gcccggggactctgctttttctctgtccttttttctc cttagtgggccatgtcctgtggtgttgtcttagccag ttgtttaagggagtgttgcagctttatgattaagagc atggtctttccttgcaaactgcttggtttagaagcct ggctccaccacttagcggctctgtgacctcggacaca tttcttagcctttctgggcctcgctcttcttcctcat aaagtgaaaatgaaagtagacaaagccttctctgtct ggctactgagaggatggagtgatttcatacacataaa gcacttaaaataatgtctggcatatgatacatgctca ataaatgtcacttacatttgctattattattactctg ccatgatcgtagcttaagaacagaggtctttacagga attcaggctgttcttgaatctggcttgctcagcttaa tatggtaattgctttgccacagactggtcttcctctc cttcacccaaagccttagggggtgaacgatcccagtt tcaacctattctgttggcaggctaacatggagatggc accatcttagctctgctgcaggtggggagccagattc acccagctttgctcccagatacagctccccaagcatt tatatgctgaaactccatcccaagagcagtctacatg gtacactcccccatccatctctccaaatttggctgct tctacttaggctctctgtgcagcaattcacctgaaat atctcttccacgatacagtcaagggcagtgacctacc tgttccaccttcccttcctcagccatttttcttcttt gtacataatcaagatcaggaactctcataagctgtgg tcctcattttgtcaatctaatttcacagcctcttggc acatgaagctgtcctctctctcctttctgcctactgc ccatgagcagttgtgacactgccacatttctccttta acgacccagcctgctgaatagctgcatttggaatgtt ttcaatttttgttaatttatttatttcatcttttttt tttttttttttttttttttttttagggccgcacccat gggatatggaggttcccaggctagggatccaatggga gctgtagctgctggcctacaccacagccacagcaatg cacaattcgagccaatctttgacctacaccagagctc acggcaacactggattcttaacccactgattgaggcc agggatcaaactctcgtcctcatagatacgagtcaga tcgttaacctctgagccatgatagttgttagttactc attgatgagaaaggaagtgtcacaaaatatcctccat aagtcgaagtttgaatatgttttctgccttgttacta gaaaagagcattaaaaattcttgattggaatgaagct tggaaaaaatcagcatagtttactgatatataagtga aaatagaccttgttagtttaaaccatctgatatttct ggtggaagacatatttgtctgtaaaaaaaaaaaatct tgaacctgtttaaaaaaaaaacttgactggaaacact accaaaatatgggagttcctactgggacacagcagaa atgaatctaactagtatccatgaggacacaggtttga tgcctggcctcgctaagtgggttaaggatatggtgtt gctgcagctccaattcaacccctatcctgggaacccc catatgccaccctaaaaagcaaaaagaaaggtgctgc cctaaaaagcaaaaagaaagaaagaaagacagccaga cagactaccaaatatggagaggaaatggaacttttag gccctatctccaactatcacatccctatcaccgtctg gtaagaaatggaaaaaatattactaagcctcctttgt tgctacaattaatctgattctcattctgaagcagtgt tgccagagttaacaaataaaaatgcaaagctgggtag ttaaatttgaattacagataaacaaattcagtatatg ttcaatatcgtgtaagacgttttaaaataatttttat ttatctgaaatttatatttttcctgtattttatctgg caaccatgatcagaaatctttaaacaatcaggaagtc ttttttcttagacaaatgaaaatttgagttgatctta ggtttagtacactatactaggggccaagggttatagt gtgactattaaatcacagataatctttattactacat tatttccttatactggccccacttggatcttacccag cttagcttttgtatgagagtcatccttaaagatgact ttattctttaaaaaaaaaaacaaattttaagggctgc acccatagcatatagaagttcctaggctagcggtcaa attagagctgcagctgccagcctatgccacagccaca gcaatgccagatctgagctgcatctgtgacctacact gcagcttgcagcaatgctggatccttaacccattgaa caatgccagggattgaacacacatcctcatggatact gctcaggttcctaacctgctgagccacagttggaact ccaaagcagactttattctgatggctctgctgatctc taacacgttattttgtgccatggtgtttatcttcact ttactcaagtcagggaaacacgaagagtctcatacag gataaacccaaggagaaatgtgcaaagtcacatacaa atcaaactgacaaaaatcaaatacaaggaaaaaatat cttcactttcaaaatcacctactgatgatgagtttat atttccttggatatttgaatattagctatttttttcc tttcatgagttttgtgttcaaccaactacagtcgttt actttgatcacagaataatgcatttaagccttaaata gattaatatttattttcaccatttcataaacctaagt acaatttccatccagGTCTGTTAAATGCACAAAACAC AACTGGAAGTTAGATGTAAGCAGCATGAAGTATATCA ATCCTCCTGGAAGCTTCTGTCAAGACGAACTGGgtaa ataccatcaatactgatcaatgttttctgctgttact gtcattggggtccctcttgtcaacttgtttccaatct cattagaagccttggatgcattctgattttaaactga ggtattttaaaagtaaccatcactgaaaattctaggc aagttttctctaaaaaatcccttcattcattcatttg ttcagtaagtatttgatgagaccttaccatgtgtaaa cattgcactaggtattaagaaatacaaagatggataa gatagagtcggcgtaaatgagatgatataatgagacg ttataatgaaactcacaattccagttgggaaataaag tccttcaaattccatgactctttctggcacacgttag aggctacagcttctgtgtgattctcatgctggctcca cttccactttttccttcttcctactcaagaaagccta tagaaatatgagtaagaagggcttaatcataggaata aatttgtctctgttctaagtgattaaaaatgtcttta tcagtataaaaagttacttgggaagattcttaaaact gcttttacacactgttctagaatgactgttatataaa taaaaaagtagatttgatctaacacaattaaatgacc tttggaaatattgactaattctcaccttgcccctcaa agggatgcctgaaccatttccttcttttgccagaaag cccccaccctttgtctgttgacctagcctaggaaatc ttcagatcacgttgttagcacgaactggttacatgtg ctgtacaaatactatttaattcatctgattaaaaaaa aagagataagaagcaaaagtttgactatcttaaactg tttgcgtaggtgagaggacaattgaccatctacttta tgagtatgtaacccagaaacttaaagctccttaaggg agctaagtcttttggataagacctatagtgagacctt ttagcaaaatggttaagactgaatggagctcactagc gtgggttcatatcctgatgctcaaacacgcaattaaa tgactttaggtgggttagtctctgttccttagtttcc tcaatgggagataatattggtagtagcgattttactg ggttgttgaaagaacatctgttaaatgttcagaacgt gttacgacagagtacagagtaatgatttgcttgtata tgtatgactcaaatagtctgccatatgccttgtgact gggtcctgtggagcaggaaggagggatttcccaccca gcagaaagttgggtaaactggaaaatagactgaggcc aggaaatgatgcaaagcgttgatgttcactgccacgg caggtgaagggcagggccagagttgtcagtagggtca ggggaggactggaaataaccaagacccactgcacttt tcagcctttgctccagtaaggtaatgtgtgagagtag aaaattttgttaacagaacccacttttcagtacagtg ctaccaatactgtagtgatttcataccacatcccaag aaagaaaaagatggctcaatcccatgtgagctgagat tatttggttttattgttaaataaatagcattgtgtgg tcatcattaaaaaaggtagatgttaggaaagtagaag gaagaagactctcacctacattttcatcactgttttg gtatctgccagttgtcaccttggtccccttccccgcc tctcccctgcctcctcttcctccttctcctttttttg gaatacaattcaggtaccataaaatttacccttttag agtgtttgactcaatggtttttagtattttcacatgt tgtgctattactatcactatataattccaggtcattc acatcaccccccaaagaaaccttctaactattagcag tccattcccttcttccctcagcccctggcaaccacta atctacttactgtctccatggatgttcctatattgaa tcaagctagcataaaccccacttgctcatggtcataa ttcttttttatagtgctaaattacatttgctaatatt caattaaggatttctatgtccatattcataaggaata ttggtgtgtagttttctctttgtgatatcttgtctgg ttgggggatcagagtaataattactgctctcatagaa tgaattgagaagtgttccctccttttctatttattgg aagagtttgtgaagtatattggtattgattcttcttt aaacatttggtcagattcaccagtgaagccatctggg ccatggctaatctttgtgaaaagttttttgattacta attaaatctctttaatttgttatgggtctgctcctca gacgttctagttcttcttgagtcagttttgttcattt gtttcttcctaggactttctccctttcatttggatta tttagattgatagtaatatcccccttttaattcctgg ctgtagtaatttgggtcttttctcttttttcttggtc agtttagctaaaggtttgtaattgtattaatcttttc aaataactaacttttttgttttgtttgttttttgttt tttgttttttgttttttgtttttttttgctttttaag gctgcacctgaggcatatggaagttctcaggctagag gtctaatcggagctacagctgctggcctataccacaa ccatagcaatgccagattcaagctgcatctgcgacct acaccacaactcggccagggatcacacccgcaacctc atggttcctagtcggatttgttaaccactgtgccacg acgggaactcccgcccattttttttaacacctcatac tttaacataaagatgggcttcacatggactgatagct caaatgaggaaggtaagactatgaaagtaatggaaga aatgtagactatttttgtgacctagagattactgata cttcttgacttttcaaacaatacttcaaaagtacagc ccaaagggaaaaaagaaagaaaaaagaaacacacata tacacaaacctagtgaataagatatcatcgatacact acagatttctatgaactggaagaccccatggacaaag ttaaagaacatatgatagtttgagtgattattttgca atatttacaaccaatgagggaatattatccagcttat aggaggaagtaatgcaaatcgacaagaaaaagatagg aaacccaatataaaaattaagaaaatacaaaaattaa gaaaggatatgaactagcattttacaaaagaaaaatc tccaaaagtcaatcagcacatgaaaatatgctcaaac ctattaattattagaaaactacagactgaagcaatga ggtgctttactttacatctttttgactgataaaaagt tagaaacaaaggtgatatcaaatgtcagggataaaag gatatagaaatcgtcatgcctgtggtgggagtatggc cggtgcagtcatgtgggaaggtaatctgacagtggtt aggcagagcaggtttatgaatacactgtggcccatca atcccacgcctgtttatgtaccaaagaaatcctgttg tggcagaatctatgggtccacccctgggagcatgaat taataaaatgtggcaccagggtgtgtgaaactccagc tagagatgagatgtccacatggcaacatgaatgcatc ttagaacatagatttgagtgaaaaagagtaagaaaca gccgggaaacccaataccatttataaaaattaaagat gcacacatacaatgtagtaaatattttgcatgaactt tcaaatggttgcctacagggggggagagtaaagaaga gtagaaaacaaagataagggagtaagtaagtagctct gcctggactgaatataatgtgtcatgaactgagaaat atggttaacataatcctcttaacttgaggtcctaaat gaatgaatgagtccactattcatttacccattcttta atgtgtattgcattataatccatttttttagaaccaa cgaattttgttcccataactactaatcagcctgcctt ttctccctcattcccttatcagctcaggggcattcct agtttttcaaacgttcctcatttgaaccaaaaatagc atcattgtttaaattatacttgttttcaaatacgatg cttatatattccaagtgtgtttgcccattttcttagg tggtagaaatttttcattctacttttctatctactca gattttcccgttggaattatttccattgctattaaac ttagaagtcccccctgtgatatgccatttttttcata ctttttaagcacttggttgcttttctttgtgtcttta agcacctagaatacttataaccattgcacagcactgt gtatcaggcagcccttcctcttccactaatttatggt ccttctcttagactatattaaactgttatttaattag gatcctctcttcgtccttatgatttaattattatagt tttctaatatgtttttattataattcctcttcattat tcctccctattaaaaattttaatgaattccatttgtt tgttcttctagttaaatattaagtcataatccaaata acttagatgtcattagtttatgtggtcaaagtaagga taccacatctttatagatgcaggcagttggcagatgt catgattttcttcagtgcataaatgcaatatctttga gcaaggggcataaaaacttttatggtattggctttga aataatagttaagaactgcagactcagtttttcctgc ttttcttgaaaaagaacacttctaaagaaggaaaatc cttaagcatggatatcgatgtaattttctgaaagtct cctgtaattccttgggatttttgttgttgtttgttgg tcggtttttttgggtttttgtttgtttgttttgtttt gttttgttttgcttttagggctgcacctgtggcatat ggaagttcccaggctaggggtccaactggagctacag ctgccagcctactccacagccacagcaacatgggatc ctagctgcatctgtgacctaaccacagctcttggtaa tgccagattgttaacccactgagcaatgccagagatc gaatctgcctcctcatggacactagtcagattagttt ctgctgagccacaatgggaattcccaattccttgtat ttttgaactggttatgtgctagcatataattttgttt cttgaatctttgtgggttttttttttttttttttttt gtctcttgtctttttaaggctgcacccacagcatatg gaggttcccaggctagaggtcaaattggagctacagc tgccagcctacacaacaactgcagcaaagtggggccc aacttatatgacagttcgtggcaatgccggattccta acccactgagcagggccagggatcgaacctgagtttc cagtcagtttcgttaaccactgagccatgatagtaac tcctgtttgttcagtcttgaacctcctttttaattct ttattccttgagggtgaaataattgccataataatac tatcatttattacatgccttctctgtgctaggcatag tgacactttaggatttattatatcacttaatccctac aacaactctgcaaagtatgtatcataatcctatttga cagatcaggaaattgcagcccaggatgcagataatat gcatccatcacaagtgactagatatagtccctctgct attcagcagggtctcattgcctttccattccaaatgc aatagtttgcatctattgtatatgtgttttggggttt ttttgtctttttttttttttttgtcttttctggggcc tcacccttggcataggtaggttcccaggctaggggtc aaattgaagctgcagctgccagcctacaccacagcca cagcaactcgggatctgagcctcatctgcaacctaca ccaaagctcacggcaacaccggatccttaacccactg agtgaggccagagatcaaaccggcaacctcatggttc ctagtcggattcattaaccactgagccacgatgggaa ctccctaaatgcaatagtttgctctattaaccccaaa ctcccagtccatcccactccctcctcctccctcttgg caaccacaagtctgttctccatgtccatgattttctt ttctggggaaagtttcatttgtgccatttttcatttt acgggtaatttttacttcagtttcttccactagcagt tgtcttaaagtgagtataattaatattcatttggaaa atgtaagcaaaacattttttaaagggccatgcccaca gcatatgaaagtttctgggccaggggttgaatccagg ctccaagttgcagctgtgccctacactgcagctgggc aatgctggatcctttaacccactgtgcccggctaggg atcaaacctgcatttccacagctacccgagccattgc agttggattcttaacccactgcactacagtgggaact cccacaaaacattttttaatgtcctttgaataaagta ggaaagtgctcgtctttgagggcagggcggcaatgcc atttccacaaggtttgctttggcttgggacctcatct gctgtcatttagtaatgaataaaattgctgacagtaa taggattaactgtgtgtggagatagccagggttagag ataaaaacactggagaagtcaaataagttgctcgagg tcctctagctaataagctattaagtgggagagtgagg gctagaaacaggccatctgtctcccaagcacatgtcc attagtggtttgctgatagccttccagaacaacagag aggactctcaaacatggtcttgcctccctccaattga tcccctccatgtgcctcacagcgggtctttctaaaat taagttctgattttaattctcccttgctatagcactt aggtatggctttcagccgtgcaataaaaagcaggcaa gagtggctcaatcatataggaggttgtttttcttaga tcccaagcaggtaatcctgggcattatggttgttctg cgtttatcaaggagccaaattctctatcacctcctgt tctatcctcctcagtatctggctctattcttcagcat ctcaagatggcttgtgctcctccaagcatggcagtca aattccacacaagagggggaaatatgaagggcagaca gtgctggtctcctgagctgtccctccggggaaataaa tgtattccttcaagtcccgtgagacttctgaagtaga cgtctgcttacgtctcacccaccagaactatgtaaac tgcacatagtgctaggtctacatagccactcataact gccagggggtgggaaatctttaaataggtgtaccacc acacaattaggatgctaatagtaagggagaaggagag aataggttttgcgcaagccaccagcatgcctgccaca attgcttaaaattcttcattgacccctcattgccaca ggatgaaatccaaacgccttcttagttgggaatctga cctacctgtctctcccacctggttcagacaccattct ccttggtcataaaattccagtcatttgtgaacatcca gctcccccatgcctccatgcctttgcacatgctgttc ttttatcttttatgttgtccttttatcttttatccaa aagagatatcccatcatcacatctcttttgtcagccc ccaaatactttgtctttcaagttcagctggaggatta cctcctatttgaaatcagctttgtctcttacaaccaa acaaggttttccttccgagacactcccacagcacctt gaactcatctctatcaatcattcatttgattgtaatg aagttgttggtggtatgcctgtgtctctgacacatct gcgatctcatgagttccttaagtggaatgtgaatagc gggatgaacagtattggtcttcagccctcatctctgc agatgttgcttgacccaaatgagcgttgccttttatt ttgattttgctttgatttgtctactccatgtacttga gccatgcatttctgtcttagcgatgctttttaaaagt cattttttggttgattatccagatttgtccacctttg cttctagTTGTAGAAAAGGATGAAGAAAATGGAGTTT TGCTTCTAGAACTAAATCCTCCTAACCCGTGGGATTC AGAACCCAGATCTCCTGAAGATTTGGCATTTGGGGAA GTGCAGgtaaggaaatgttaaattgcaatattcttaa aaacacaaataaagctaacatatcaatttatatatat atatatatatatatttttttttttttttacatcttat attaccttgagtattcttggaagtggctagttaggac atataataaagttattctgaagtctttttttttcttt ttccatggtgagcagtggcttgatgtggatctcagct cccagacgaggcactgaacctgagccgcagtggtgaa agcaccaagttctagccactagaccaccagggaactc cctattctaaattcttgagcacattatttaggaacct caggaacttggcaggattacaggaaatatatctagat ttaaaaaaaaatcttttaacagaggtcccaaaggaga gtcatgcacagctatgggaggaagttcagaaactgcc cttgctaccagatcactgtcagataaaatggccagct acatgtttctgcacattgccctaagatctttacaaac ttttctgtgcatttttccacttttaaaagaaaatttc ggggttcctgttgttgctcagtggttaacgaacccaa ctagtatccatggggacaggggttcgagccctggcct cactcagtgggttaagaatctggcattgctgtggctg tggcgtaggctggcggctacagctcagattggacccc tagcctgagaacctccatatgccgcaggtatggccct aaaaaaaaaaaaaaagagagagagagaatttcctcca gaaaaaacactttggtagtttgggagaagtaaacaac caaaaattaatttttctggagtattcgggaagcttgt aaaaatgggctcttacttttttgaggagacaaatggg aacctacccagaagaggcacaatcacctgcatttgat ttcttgacctctccctaccttctttgctggctttcca catttggatttctgtgaccttatctctgctccttggt gttttcatttttcctgtggacgtgccagactatggga agggagtaaggcgttgatttagaatcctgtagtctct gcctgtctctagtcattgttttcacccttctcaaagg accttgacatcctgagtgagtccgcaagtaatttagg ggagaagccttagaagccagtgcagccaggctacatg actgtgtccacccactggaaccagtcatttttatacc tattcacagcccccctaccatttaaatccccagaggt ctgccataacatctgtaactccctttcctggtaaatt gtgttctaaaagactggtaacaaaagatattctgtgg tacagagcataattaaatacctgggagctgatttgag tggggtaaatcaactggtttgacccctaaaacccacc atgagcatttctgttcaataaagtaatgcccgtgctg ggaattgtgttctacggaaatgctcctgctgtgtctt tcttgagtcctgtgtcattgaacatgcttaggagcaa aggtcccccatgtggcttgtctgctaaccagcccagt tccttgttctggctggtaatgatccgatcatctgaat ctcactgtcttccaacagATCACGTACCTTACTCACG CCTGCATGGACCTCAAGCTGGGGGACAAGAGAATGGT GTTCGACCCTTGGTTATCGGTCCTGCTTTTGCGCGAG GATGGTGGTTACTACACGAGCCTCCATCTGATTGGCT GGAGAGGCTGAGCCGCGCAGACTTAATTTACATCAGT CACATGCACTCAGACCACCTGAGgtaaggaagggtga gccctcaactccgaagaaaatgctgcaataaaagcac tgttggttttcagctttttttgtaatcactgctcatt ctgaggtagattcgcttgggctgataaaaagagaact aattcagataaatgcttgcatttgcatagcctctttt tttaaaaactttttttttttttttttttttttggctt ttcagggctgaacctgtggcatatggaggttcccagg ctaggggtcgaatcagagctgtagccccgggcctatg ccactgccatagcaacatgcatagcctcctttttaaa gtgccttcctgttttataccattgggatgtgagaaga gctattgtggaaangagcatggggtnataaccctgga cctctcacgtcctaccctcaggntagtgggaaaactc tgagtttaaggacatcaaagtgactcctttttagtta cattatggnggaatcagcncatatttttacaaggggc ggagngtaanctgttggagtttacaagacatatggtg gcattgcaactacttaaccctactattatagcacaaa agcagccatagtcggtcctgaaggagcctgatgcctt cagctttataggcaatgacgtgtgaatatcacaaaca gtttcctgtgtcaccaaacatgattgccttttgattt ccctttcaaccctttaaaaaaaggtaaaagcccttct tagcattcagcagcaggtcgctgtgttttgccaactc ctgatctgtagcatttcgacaacactgagctctcaac ttttgaaccctgagtccaccacatccttcagtgaaac cagagccatgtgatactaaggatagaaacggaaactt cctgaatccaggcgatcaaataggagggagaaagagg aactttcattgacaaaaccacaaatattgtgaatgga ctgttacaaatattgtgaatgctcctattcccaaccc cctggcttcattacagggtcctatgtgttcatcctta ttgagaaatttgtattgctactgccaggttgccaata cccagcggtgcccatggtgttctaaaatgaagcaatt tcaactttatttttttttcctgtgactttacatgaca agttcacatgaaggatatactttgatagtaatgtcca tggttagggaatatacattgtttgctggttgactggc ccctggatttttctattgaaagtccatgagatctcga aggcacaggtgtgttctctcgctttttaaggaaaggg tttaaaaacttaagtaattaacagctttagtaacaaa ttacctataacacacttaaaaaccgaataccacccac tggagtattgtgctacgattaaaaatctacttgtcta ctacatgatatctttgtcccacagaaggttctggaac caaacttgtaatttcaggattatgagagccctgagtt cacgcattgtgtaataactatgttgtgtggtagtcaa tttgtacagcttgcttagagagaacaatgtcaagtta aggaggcgattgctttatagtgcctgtcacaagatgc cattgccattgtcctagcaagagatattctatgggag tatactacattttagtgaggataagaactttttatgg catttagtccggtcatttcccaaccactgtcctgaaa accaatttcattttgatttcaggggcttgtgtgggca aagttgccaggcattaaaaagccacttctcaactgta gtatcacaatgctttagttgggtagtgtattgcagat agcttatggctgaaaagttaccaagccttgcagtttt cactcctttgagtttatttccttgacagaattgaccc tgagttttttgactcttacctgctcaactaataaaca ccagagtcatttatctccattgctcttgtctgacctt tatttaccgaataatgccttatgggttcacaaaaaca aggggggagggggccagcatgccttagaaactgtctt tagtcaagaaatgngattttattatgtaaatatatga gtattataatagatagtgttattaatagacaccagca agaattgtcaataatttaaaaatcacaaattaaaata catccatgttagnatcatttatcctaactcccaaagc cctttaaagtggaagatttagatgttaacccagagat taaagacatgttcaaagaatccttgatttttttttga atcccttgtttttagagaagaaaacctaatgattttc cccctctggattctacatattaaatatagttttggaa cttgaatattagtatggttaataagtgctgatatgct gattttgtttatatttttcttatgagtaaatatccta tatcaccagacattatagtctatgtacaaatatgatt cttaaacctgatagcacattcattagagttggaattg ccttttttttttttttttttacagttgcacctgcaac atatgaaagttcccaggctaggggttgaatccaagct gcagctgccaccctacattacagccgtagtaacagca gatccgagctgcatctgcaacctatgctgcagctcag ggcaatgccagatccactgagtgaagccagggatgga acttgcatcctcatagagacaacgtcgtgtccttaac ccactgagccagaacaggaactccagaatttcctttc aatagaagaagcaccaagtttaggatcagaaagcctg aatttgaataccaatttactattttagtcatatattt ctgagtgtgntcctcatttattaaaagcagactaaaa gatgagagggtcttttgttgagaatcaaatacaataa catgtgaaagtgtgtaacactatgattgaaatatacc tacacagccatttatttgtttattgttcatgttttgc cacccacacagtagtatataatccttttatgtaataa atgctaataatgaaagttggcaacttatgtaagtact caaaatgctggaggtcatgggatactgactgggatac tacagaggtaatgtcatttcctctgcgctaaacttat tgtcttgtagttagggactgactctctttaggacaag gagttcattctgtataccatgtgtggctatcaccctt cgaagttgaaaaactgccccagggtgggcacccatcc gttctcttagatatatggccgagacctttctctcact gggagggaaccacactgaggaatgagaaaaaaaaaag gaaaatcaagatgaaaccagaaacctctttggcataa cttctccactctgtactttttgttagaactacccttg cacaaagcagcatcagtgtggaagacagaatttgcac acctggtttgatatacatgccgtggtatatgggatgt tctaacaataaagaggactctcccaggaaatctcctc actgttatagtcagccttgaggaaagagctcttcttt tggactctggggagagtctagtttttcagttccttgc ttctcggtcaacgtgttggtgtaaggatcacactctc tcttatactagataattctattttttcaccTTTcaac ctgtctatccttctgaccctagTTACCCAACACTGAA GAAGCTTGCTGAGAGAAGACCAGATGTTCCCATTTAT GTTGGCAACACGGAAAGACCTGTATTTTGGAATCTGA ATCAGAGTGGCGTCCAGTTGACTAATATCAATGTAGT GCCATTTGGAATATGGCAGCAGgtctgtgttctttcc acatgtttgggttatcctttctgggataaatttgagg cgagatagaaactttaagactaaagaaacaatggcct actttttttgtacatggtcctgtgtaaatctctattt gagctgaaataagatggtcttcctctccaattatcca tggtatgactctgatggataacaaatccagttctgaa aaaaggggatttctttccagaagagaggacagtttct tcaaatattgaattaaaagcaaaatagatgtaaaccg ttgttggttttattgttgaattccagGTAGACAAAAA TCTTCGATTCATGATCTTGATGGATGGCGTTCATCCT GAGATGGACACTTGCATTATTGTGGAATACAAAGgta ttttcttgccctcatcagcatgaaattgctcttggta gaaaggataataatagttatccaaaacatcatcctat gttcatctgtttcttccctcttcattttccatagagt acagtatattctatctctgtcttaggaaaatggactg tcattcatataatcttacagagaatcaattagtaatg tactctatgccgtgacaggtgcgaaggttttttttga aggcaacagataaaaatatcctatatttcacctattg taatttccttaaaactgacattattgaataaatgttt tactttcatcttgaatattattatgttatggaatcat acactttaccccaataatcatcgaaaagaatttccaa aaggttgagagagttgtgttgatctgattactttcct ctgcatcctttgagcttaacctttgaatatagtttgc taaggaaagtagtctgtttatgatcctggagtggaat caggctaagtgtcctcattcagaacccactgaatcag acagaatgaatttatttccttgaaagttcaaaatgtg tcactcaagagtataaattttcaaatcttactctctc ttttccttggatgtgagcaattcttcgataattgaat gaggcagattatatagacttacatggaagactgttgg cctgagaattcaaactatggtgttcaagacttcacng ngagtccgatgccatttgtttcccacagGTCATAAAA TACTCAATACAGTGGATTGCACCAGACCCAATGGAGG AAGGCTGCCTATGAAGGTTGCATTAATGATGAGTGAT TTTGCTGGAGGAGCTTCAGGCTTTCCAATGACTTTCA GTGGTGGAAAATTTACTGgtaattctttatatcaaaa tgatgccaaggagttggcatggcactttgctaaatgc tgtgtgaatcaatacaaagataattaggacatggttc ttcctcacaagaggtgtgcaatcttattgggaaatca tacttgcaagtcacaaatatagactaaagtttccagc tgagaatatgctgatggagcatgaaacactaaggaga cagggagaatctcaggaaaaatcaagaataatttgga tcaaatggattcctgacatagaacatagagctgatca gaaagagtctgacattggtaatccaggcttaagtgct ctttgtatgtggttcagaacagagtgtgggcagcctg agggggatacatacccttgacctcgtggaaagctcat acgggggagggatgaggctaaggaagcccctctaaag tgtgggattacgagaggttgggggggtggtagggaaa atagtggtcaaagagtataaacttccagttacaagat gaataaattctaggggtataataacagcatggcacta tagatagcatattgtactatatactggaagtgctgag agtagatcttacatgttctaaccacacacacacacac acacacacacacaccacacacacacaccacacacaca cacgtgcacacaaacagaaatggtaattatgtgaggt gatggcggtgttaactaactttattgtggtcatcatt tagccatacatgcatgtcatgaaatcaccatgttgta caccttaaagttatgtaatactagatgtcagttatat ctcaaagctagaaaaaatgtggggaccaaggcagaag ctcttctgctctgtgtctaagggtggttctggggctg ggatggggaggatggttaagtggtatatttttttcat acctttgctcagtactatcattgtaagtgttcaatat atgtctgcttaataaattaatgtttttagtaagtaat ctctgtttagtaatgtgtcagaaatgccctacttgca ataggaagaaaacctgtccagtcccttccttttttct gtaagtctgatttcattgcctcccagaatgcatcacc atgtgagagatagagggaaggtgctgtccttatgggg ttaacagtgtgactagggaggcaaaatatacctacta aagggtggtagcataattcagttcttatgtgagtatg tgtatgtgtgtgagtatgtgcacatgcacatacattt taaaaggtctgtaatatactaacatgttcatagtggt tacacctagcttataggtaacattttttcccctgtat ccttgtttgtgtttatcaaattttcataacagtaatg gtagaaggagtacctgacatggtaccatacatgctng gncctgcctaatttctcnatttcctttattgcccata cccccattgcttgacaagcataagtccatactggctt gttttcgttcctcagactcagtacaccatgtagctcc atgccctgggtctttgtatgtgctatttctactgctt agagtgctattgcccctgaccaccacgtggtcagcaa cttctcttctgcgtctgtgtctatggtctatgattcc agatgtcatcttcactaactacccttctaatatgccc ttccatcccacccgtcctcatccttaccccagccact ctctatttggtggctctgttttattttcttcctagct catcactctttgaaatgaacttatttacttattcaat tgcttctttcactagaatgaatgctccatgagagcag ggacctgctttatcttgctcgccactgtattcacagt gcctagaactacgtctggcacatagtaggtgctcaat aaatatcgatcaaatgaaagaatgagcaaacgaacaa atgaacaacacgtgaggtaggcatcatgattccatca acagaggagaaaaccagacttaaagnaatgaagtggn ggagctgcatttgatcttgactgactccacatccatg ctcttgaccactgtgcatctccagagtgtaatgaaca tactttacttttatattccaccaaaataacaaagcca tgcccatgttagtagagagttaatcgacagtgccctt aaaatatgcatgcacccagggtacaactatgcatgct gccctgtgttttcagttggatccaaatgaattgccgt aaacaaagaggggattcaatgtctttgactagtttgg gatattttcctagtaaccaactttgcaaaataaagcc actaatgacaaggagctttgttctacttctgcatcac tcaactgtcaatttttatctcttgcaagacttctaat ctactagaacttttgtttttctgtgatttctgaacag agaagactaatccaaaccctgtcattccagAGGAATG GAAAGCCCAATTCATTAAAACAGAAAGGAAGAAACTC CTGAACTACAAGGCTCGGCTGGTGAAGGACCTACAAC CCAGAATTTACTGCCCCTTTCCTGGGTATTTCGTGGA ATCCCACCCAGCAGACAAgtatggctggatattttat ataacgtgtttacgcataagttaatatatgctgaatg agtgatttagctgtgaaacaacatgaaatgagaaaga atgattagtaggggtctggagcttattttaacaagca gcctgaaaacagagagtatgaataaaaaaaattaaat acaagagtgtgctattaccaattatgtataatagtct tgtacatctaacttcaattccaatcactatatgctta tactaaaaaacgaagtatagagtcaaccttctttgac taacagctcttccctagtcagggacattagctcaagt atagtctttatttttcctggggtaagaaaagaaggat tgggaagtaggaatgcaaagaaataaaaaataattct gtcattgttcaaataagaatgtcatctgaaaataaac tgccttacatgggaatgctcttatttgtcagGTATAT TAAGGAAACAAACATCAAAAATGACCCAAATGAACTC AACAATCTTATCAAGAAGAATTCTGAGGTGGTAACCT GGACCCCAAGACCTGGAGCCACTCTTGATCTGGGTAG GATGCTAAAGGACCCAACAGACAGgtttgacttgaat atttacagggaacaaaaatgatttctgaattttttca tgtttatgagaaaataaagggcatacctatggcctct tggcaggtccctgtttgtaggaatattaagtttttct tgactagcatcctgagcttgtcatgcattaagatcta cacaccaccctttaaagtgggagtcttactgtataaa ataaactattaaataagtatctttcaactctggggtg gggggggagactgagttttttcacagtcctatataat aattttcttatcctataaaataattaggagttcccgt agtggctcagcaatagcaaacccgactagtatcgatg aggatgcgggttcgattcctggcccccctcagtgggt taaggatctggcattgccgtgagctgtggtgtaggtg gcagacacggctcagatcccacgttactgtggctgtg gcataggccagcagctccagctctgattagaccctta gcctgggaacttccatatgctgtgggtgtggccttga aaaaaaataaataaataagataattactcaaatgttt tccttgtctcagaaccttacttcaggataaagagtga gaaagttttttttatgaagggccattattacagctca aaaataagttgtcttcagcaagtagaaagcaataagc ctgagagttagtgttcctatcagtgtaaatattacct cctcgccaatccccagacagtccatttgaacaattaa cggtgccctgggagtacagttcagaaacattaatgtg gatgttccagacctgtatttttataagtacttgtctt gagccggatggaaccatcattcctcaccattatttag aagtggactgtgactctgttggagatcagggcacacg gttaccaaaagcacacccttctcctggccttaccttt gcaaagctggggtctgggacacagtcagctgattata cccttttactaacttcccacagctcaaatctggtcaa ttctccttcacaaatctcttaaaaatccatcactcac ctccagcctcttctgctgtggccttgattcagcctct cacaatttttttttaaccagaattctggcagtggccc ctgacttgcctctgtgctcccagccccgctgtcctct gatccatcctccatgccagccttcaatctgctggtca cgattcattgatgggttaggaaatcaatggcatcaca actagcatttagaaaaaggaaataggcgttcccgccg tggcacagcagaaataaatccgactaggaaccataag gttgcgggttcaacccctggccttgttcagtgggtta aggatccggcattgccgtgggctgttttgtaagtcac agacatggctctgatccggcattgctgtggctctggc gtaggcctgcagcatcagctccaattagacccctatc ctgggagcctccatatgctgcaagtgcagccctaaaa aaaataaaaaaataaaaaaaaataaataaaagaagta gacaaattgtatagaacaaccctgagtatgttgcctg agcacatataacaagggtaagtattatttcaggaaac tctggtttcacagatactcttggcatatggaccccta gagtcctgatgtaaaatatattcttcctgggatctta ggcaagaagtttgaaagctccaactctgcactgctgc caaagaaatgatttttaagtgcaaaactcttcccgtt cccttccctgtataaaattccataggatctctccagt gcctctaggataaaggcagttttcattctctagttca aggtgagagaagattttaattatttcacgttttagtg gggaattcaagagtctggcacctgacatttgctgaac tctctccattatccctctctagttccccagacgcatc ctatggtagaaattcgcaactagagtgagcgtcagag taacccaaggaaactgggtaaatgcagctccctgggc tctaccccctgagattctgattcagtagatctgaagc agagccctggaatatgcatatgcatcattgtgtcaca ccaagcattctgggtaatgagagttgatgttaggttc tcagtagtaagacaagtatagagattccgggggactg agtgctcagctctgccttggggaggagggagagggct aaagagaacaggagatggggacagggaatgctcaacc tccaatcttaggcatttgagctatgtcttaggggtca ggaggaggttaccaatatagtgattaagagattgagg ttccagtcagagggatatgctggagaaggggggtgaa aataatgtcataggtttggtgagtgcagatactttga gttttttaatatttttattgaaatatagttgatttac aatgctcttagtgagtacaattactttgaataagtgc atagatgtatgccattcttccagaaatgatttattga gctcctttgggcatcatgctaagtacaggggaaacag ctgtgaagaggtccttcccttatgaagtcattcatcc ccttcagtaaatgaaggtaaaggaaaaggatgagaca gggacgccgtgttggaccagggtcagaaaggccttat aagaccttgcctggagggcaaggaacttgcctgtgag taaggagagcttgagaaagcgataaagcaaagaagga acattactgcattgtgttttagaaaaaccatgtcctg gggaagaactcctagagtcaggggggccagttgggag actgtgcttttttccaggaggagataagtgaggctgc tggctgagatggagcaaggatttagagaagcagatat gagattcatttagaagttagacattttaggatctgac acataatttatcaccaaaaccagtgcatctctggctt tgggccaccagttttggagaagtggaatgtagggacc taccattacctgccaatctttactacacagatgccta tttccctcctcatatttcctttctccagatcacgtcc tattctattgccaggactcaagattccaccttgcatg cagtgatccatcttcacactggatggacagctctagg gatgtcagagcacactcttgtccatactgctgactgg gtctcctgtcagcccatctgtctatcagctgtggtat tattagtataataagagggctgtatatgagagacaca aaattctaggtgtagctcaaagataggctagagttat tcctatgtacaacaaatatttatgggaccccttctgt gtactgtcatggttgctgctttcatcatacttgtagt ctaatggaggtgggggcagggcaggaataagcggatg tccacaaaatcagtaagaccacttatattcaacattt tcataatttagttatttgagcccaaagggtccacatc cgtggtattccaacttttttttccccggacatggatc tttatctttttttttttttcttttttgcggccagacc tgcggcatatggaagttcccaggccaggggttgaatg ggagttgcagctgcctggtctacaccacagccacagc aaggtgggatctgagctgcatctgtgacatacaccgc agctgaggtaacaccagattctgaacccactgaatga ggccagggatggaacccgtctccttatgaacactatg tcatgttcttcaccctctgagccacaacgggaactcc agacttcgtctttaaatgtattctgacttggagagct atcacactaagcaattaacaggagctgacctggttta ggctggggtggggccctactcctcaatgttccctgag gcacatctgtgggacccctgggcatcatctatctgag cagccttagagctgctcatccagttgactgttgatgt agaagtgcaaacttctgccttccttatttgttgcttt cttttttcattgttctctcccctttgtgtctttaagC AAGGGCATCGTAGAGCCTCCAGAAGGGACTAAGATTT ACAAGGATTCCTGGGATTTTGGCCCATATTTGAATAT CTTGAATGCTGCTATAGGAGATGAAATATTTCGTCAC TCATCCTGGATAAAAGAATACTTCACTTGGGCTGGAT TTAAGGATTATAACCTGGTGGTCAGGgtatgctatga agttattatttgtttttgttttcttgtattacagagc tatatgaaaacctcttagtattccagttggtttctca ataagcattcattgagccttactgactgtcagacgga gggcgtattggactatgtgctgaaacaatcctttgtt gaaaatgtagggaatgttgaaaatgtagggaatgaaa tgtagatccagctctgtttctcttttggaggattctt tttcctccatcaccgtgtcttggttcttgtttgtttt gggtttttgtgggtgttgtattgtgttgtgttggtta tggcagtgacagctatttaaactgtgaaacgggggag ttcccgtcgtggcgcagtggttaacgaatccgactgg gaaccatgaggttgcgggttcggtccctgcccttgct cagtgggttaacgatccggcgttgccgtgagctgtgg tgtaggttgcagacacggctcggatcccgcgttgctg tggctctagcgtaggccagcggctacagctccgattg gacccctagcctgggaacctccatatgccgcaggagc ggcccaaagaaatagcaaaaagacaaaataaataaat aaataaataagtaagtaaaataaactgtgaaacgggg agttcccttcatggctcagcagttaacaaacccagct aggatccatgaggatgtaggttcgatccctggccttg ctcagtgggttaagaatccagcgttgctgtgagctgt gatgtaggtcgcagatgcagcccagatcctgcattgc tgtggctgtggcgtaggctggcagctgaagctccgat tcaacccctagcctgggaacatccatatgctgcaggt gtggccttaagaggcaaaaaaataaaaaaataaaaaa taaataaattgtgggacagacaggtggctccactgca gagctggtgtcctgtagcagcctggaagcaggtaagg taaggactgcagctgggtaaggactgaattgcaccaa ctgggaagtaagcctagatctagaacttaagttagcc ctgacatagacacacagagctcaccagctaagtggtt cagcttataagctggtcactgaaactgaggatgtcca caaaagcaaaataagtagcaacaggcagcgggatgca agagaaagaggaggcctaaaatggtctgggaatccct gccatacctatattttatcctacttatatttagtgcc tgaatgtgtgcctggagagcaaagtttagggaaagca tcgggaaatgcacagtattcatacccttaggaacaaa gatcagttacctccagggtaaagactatttccaagtt taaatttcaacccctgaacattagtactgggtaccag gcaacacttgccatcctcaaaatcaatgaatcctaaa attcaacctgggggtcagtgacagtctgtgacaaagt ttttgctggtcagtaacgaaataagtatgagcaccat ctgagtatggtcaccaagatgtcaactctctttcctt tggacgaattgtcattattccaagattaggtcctttc tatttttgaggtgtgaaaacatctttcctttcataaa ataaaaggatagtaggtggaagaattttttttgtttt ttggtctttttgctatttctttgggccgcttctgcag catatggaggttcccaggccaggggtcgaatcggagc tttagccaccggcccacgccagagccacagcaacacg ggatccaagccgcatctgcagcctacaccacagctca cggcaatgccggatcgttaacccactgagcaagggca gggaccgaacccgcaacctcatggttcctagtcggat tcgttaaccactgcgccacgacgggaactcctaatga tactcttttatatttagctactatgtgatgatgagaa acagtccacattttattattttttagccaatttgata tctcattactaagataatgataattttctctataaat tttatttaagttagtgttatgaagtggttttgctagt gtagaaggctaggatttgaattcagttcaagaaagaa gagagggagggagggagagggatgggtagagggatgg ggcagtgggagagagcaaagaggagagacagtttttg tattaattctgcttcattgctatcatttaagggcact tgggtcttgcacattctagaattttctaaggaccttg accgccagattgatatgcttcttccctttaccatgtt gtcatttgaacagATGATTGAGACAGATGAGGACTTC AGCCCTTTGCCTGGAGGATATGACTATTTGGTTGACT TTCTGGATTTATCCTTTCCAAAAGAAAGACCAAGCCG GGAACATCCATATGAGGAAgtaagcaggaataccagt ggaagtgcccctttcttccttccttcctaaataaact tttttattttggaacaactttagagttacagaaaagt tgcaaagatattatagacagtagtgtttatatatata tataaatttttttttgctttttatgaccacacctgtg gcatatggaggttcccagtctaggggttgaattggag ctacagctgccagtctgtgccataaccacagcaatgc aggatctgggccacgtctgtgacctacaccaaagctc acagctggattcttaacccactgagcaaggccaggga ttgaacctgcatcctcgtggttcctagttggattcgt ttccgctttgccgcaatgggaactccaaattattgtt aatatcttactttactggggtacatttgttacaacca atactctgatactgaaacattactgttaactccgtac ttgcttctttttgagtcatttgcaaagactggcttct tgacctgcttccttccaaacagctggcctgcctatgc tgttctcagacctgcaagcactgatctctgcccccct tgccttctctccagtggtgtctccttccccaaacaaa cccagtgtggctctggaaagggagttaagtcaacata aaccaacacatattttgttgagctccaattttgagca aatccctcaccacggcagacaggcatgatgttaagaa ctagggctttggacacaaggtcaagaccaagaagggt tcctcacccctactgattcagataaccaataatgagg ctttgaatccctgtccaaaggttgttttttttccctt ctattgagcttcttgccaccttatcagttttttttat gacagtcaaatgacatgatatatgtgagcatacatgg taatttttaattctatataaatgaatcactaaataaa ttaggaggatatatagtccacctttaagcgtattaca cgtgtcacatgaatgtgtggcgacttaattgtagagg tttaaatgtagcttcctataatagatgtgttcctaaa ctacattttaatcattggacttgtatttttatgttag cacttgctgttgaagaaaagcctatgccaaaagttca gtgaaaccaataatccactgccagctttctgagttaa aaaaaatccctgggttttcacacacaggaacaccctg tgtgaaacactcatttagagcaaaatgcatctgataa ggagttcctgttgtgcctcaactggttaaggacctga cattctccatgagaatgtgagtttgatccccggcccc actcgatgggttaaggatctggtgttgccacaaactg cagctccgattcatctcctagcctagaaacttccaca gcccagaatatgccacagaattcggctgtttaaaaaa aaaaagaaaaaaaaaagaatcataaatgtgttggttt gttcaccaaatacatgataacttgctcttgccaagct cagcttcataaatattaagtcatttaatacagcagcc accttatgaacagatattactatacttcccatttaca gataaggaaaatgccatatttaaccaagagattaaat aactttcccgaggtcttatagcaagtaaatcatggtg caggggtttgaccacacgcagtctatctccagagtct gtgtatttagccactgttttactttcaaatttaaatt tataaaacttctaaattatctgttaaccataatcttt ggaatttttaaaaccacgagttcctataaaatgtttc attgaaagtaagtcacttttccatagcttttgataat acatctgtaggataaagtaagccacagctctcttgca gacttggtacaccctggggcaaagcatcatgcctgtc acgtacatggtggtccttactttgactctcagtgctt ttattgcccaggaattttgtgagatttctagttgttg aggtttgtttaaagaggttatgccggtacttggaaga gctcttttcttgctacctggagccttctcatatttcc tttttgaggagggacatgaattgcctttcaaactcat aaatatattttctagtacacaagtctccatcttcctt agacgcatggctcctggagttctccatcctcctgctc cactttgggtgggctcctctctgggtctgccaccaat ctgccacccagagacatccttgacccacttccagacc ccaccatggcttcactttcttcgctttcctcctttgt ggaaccttctgcttaagaatctgaggaagaaaatttg cacgtgagctaaactggaggtactttcctgcctggtc ttgcacgatagcttggctgagcccatgatgctgggtg gctgttactttccatggacacccgaaggcgttgctcc tttggcttctagttgcatgcagtgttgcttatcccag gctgatctttcttccactgtaggtgacttttaagaat taagggattaatctatatctacaacaacaacaacaaa gaccttttcaagctgaggtagggctttctgtatatgt ttggagtggttatccagcagactttacttgaaggcag gggtcatatcctcaagtgctcataaacggaccacaga aagatctcataattgggtggagctgggtggggaccgt gtcatgtggccaggaaatgccagatgggaagggagtg gcccttactgagctccagctgaactctgaattttcta gaaaactcagaaatctggatttttcatgtgtaatacc cagatttatagatgtggaaagctaattcttttttttt ttaagggactataggcaatgaactaagatctaggttg tatttggacaaggggtcatcagtttaagctgtgtagt tgagcgctcagctattgggctgagggacccctaaata ctgagacggggaggtccttgctctggggcatcacaag tacactccctggtctcattcaaacacttttcctacaa aattgatcccatttcttcagtgcactgtctgaatgca tttggcccagagccgtgctgaggcatagggaaggggt ccacggtttcatggcatcgttttgtgctgtgtgtccc tgctgtcgtccaggatacctacctctcctcctcctgc atctgaatgtccccccacagactctctgggattctac agcctctggcctgttcctcagacacctcttacctgcc agctttccagattcacattagttagtccaaatctact gccgtcagtgactcacttcatttcttcttctccgagg cagttcagcccggtacagttgttttgtcaacacttca gttgagtctggaagatgtgcatgggttatgcacgaga gcggtccatcattttgagctagaagtcctttctcagc ccagagacaagtcctcatctcctttacttcctgactc ttcttcctctgcatccttccaagatatctctttctcc agccaccacctaaatctcttcttttcccggggttccg tgctcaacccactcttcttcttaaatctgtggctggg tgaacgcatctgctggcaccacttctctgctaaagac tccaaaaatccataggtcctgcccggcctttgcccac ctctctccaacactgtccagctttagatgtagagcta atccccccagagatatcattccctggatgtctaagtc ctttggtatctcactttcagcgtgttcaaaatcctct tacaactgttctttctccttttccatcttgattattg gcaacatgccagcctttcccctacccccagcagtgag ccaagctagaaacaagggcttaatcttcaatctttcc ttctccatccctaaacctaatgagtctccaagccctt cccagtttacaccctaaatgttgctcaaaacatcccc tagttcttccacgtgctctcctctatattgaaaggtc aagaaaggccatcttccctccactgtgaggaaataga tcttgatactgcccctgagctgggcagtcctcgacct gacaaactgtgcagtgtttctaaatctctactggcaa aatgagagtgcctttgacctgtgttgcgatctcagat cacagtggatgtaattgttttataggaatggtgaacg aaaaagaagtaaatccctaatgccaaactcctgatca ttctatgtcatttaatagcctgtcatttatgataaag tttcctctactggcattagcacaatacttctcaggaa aaaaaaatatgatgccagatactgaaaagctcctggg taaacatgaacatgggtaccgataaaatggtgaagcc agtccaatcttagagtgacttcccttcatgctacttc atgctcttttttttttttttttttaagaaaaacccct tttttttttctcacaccagtcacagaggagaccgagg cttagcaaggttaaggtcacatgattagtaagtgctg ggctgaaactcaaaaccatctctgcttgtctcctaac cctgtgcacctctgactattcaacagATCCTGTGTCA GGAGTTGGGATTCTTTGAAGgtaagggccttgaccac cgaattaaggtaatcttgctctgtggcaggccttgtt ttcagtattttaagtacactggctcaggtaatcctca caacagccccaggaggaatgttctattacctccactg tatagatgaggaacttgaggcacagaatggttgccaa ggtcacacagctatattgggggttcatacccagccat ccaactctgtctgtactctctgccactctgcaccccc agctcctgatccacttcctgtttccatccctcgattt ctgctgcactcaggggcccctctccccctcggcctgt gagatctgcttcagtaggcttttctccctgactcctc catccctgtccttacaggcagctgcttctctccggga cacgaggggtccatacggacactctctactggctggg ttgcgcctaactcgtgattcctcctctgtttcagATT CGGAGCCGGGTTGATGTCATCAGACACGTGGTAAAGA ATGGTCTGCTCTGGGATGACTTGTACATAGGATTCCA AACCCGGCTTCAGCGGGATCCTGATATATACCATCAT CTgtaagtccgaaaatgcctgtcgtgtgtgccttagg ctgctgcggaggaggccagggctatataagcagagtc agtgactgactgtgccctgcagtgttgatggccatgg agattccaccgttagagcttttttctttgttaacctt gaaggcaaatctggttaggaagataactttcaaagag tcaccatctggacattcatgcccatgtgcttcaatcc tgtatacaagcagtttagagtacagggaagggaagga cattatgaaagggagagggtgtgtttggatccagcag ctccatcctcagaatttatctgaagacactgcaaaat tactaagaatcactatgacaagaatgaggatggggtg atatggcaaagttgtgatcctggaagaccttcatctc ccatgttgcccaactctgaacatgaatttggtgaact agttggttaaggggatgatcctccaagtttctccctg gttgagctccaaaaaccatgtaagtttctcatagcaa aaccgtataggtccttagggctttagttggaatattt gtgctgaaatgctggaaagccccatttgccatttttg tatttgcaaaataatcatcaagaggggagaatgcatt ctttcatgaccactgaccctctgaaaaggtcaggaat ttagtctgaagtaggcaagcctcctaccccgcttctg ccatgagcttgcacgcacaggcctglcttgacatttc ttctttatagatttctttttgaatatcttgaaattgc tttaaaaatatttaaagaatgtagaattatataaaat aaaaaggaaataaccccacacctcccacaaaaccctg tttcctgcctttctccacccactctccagggtaacac ttggtaacagcatagttgtatcaccccaggcctattt ttgagcatatcagcatttcaagaaatgtattttttct caataaaacatcccttatagttgaggaggggaggtta tcattcctgggttttgttttttttttttttttaatgt aatcctggtacatcggtaatttgcattttttattcat taatatctttggtatttctagtgttgggacacacagg tcaacctcagtttttgggtttttttttttgtcttttt gtctttctagggccacacctgcagcatatggacgttc ccaagctaggagtctaatcagagctgtagccaccagc ctacgtcatagccatagcaacgtcagatccaagccgt gtctgtgacctacaagcacagctcatggcaacaccgg atccttaaccactgaacgaggccaggggatcgaacac acatcctcatggatcctagtcatgttcattaaccact gagtcatgatgggaactccaacttcaactattttaat gtctgtaaaacattccatttggaaaccatttcatttg taaagcaaaatgaaaacattttgttcattttcaacag agttcgtagctgacttctgttctggaaaaaaggaaat ggagcaaatttgagtgagaaagattcaaagataactt ttcttttaaaaaaaattatatcttggaaacttctggg ctattgattctgaagactatttttctatatactgttt tgatagcaaagttcataaatgtgaaaggatcctgcga tgaatcttgggaagcagtcatagcccaatatatcttt gttgcttttaaaatgagatttagtttactaaatattt ttctgatcataaaaataacacagatctaccgcagaaa atttggaaaaaaaaaaacttttaaattcaaaaaacag ttaaaccacaaatgatcccaccatccagagagcaatt tgtactttggtgtctagttcatctttctttttctgtt tacaagcacatataccacaagcattttttcaaaaaat gaaaatgggataatactatacatacgtctgtacacct gcatagttactgaacagtctttgatctaccctgtaag tttctaacttttcattatttgaaatgatgttttggca aagaaatatgtaggtgtgtctcgcacactttcataat gatttcttaggataaatttcttaggataaattcataa tgatttcttataataatccatactctgccaactgatc ttcagggaagccaactcgccttctcagaaataacata taacccatttacttgccctctcaccaatactaggtcc taatgtttttgtgtacagattctatatttttacatac aagaattccttaaagcaaggcatgtcacagaaaaata gaaggaagacacaattgtcatgtttaaggactgcatt ctgtaccaaaaatgctaagttaaatgaacatctgaaa cagtacagaaacgctatctttcagggaaagctgagta ccaggtactgaacagattttggcaaatacagcaggca tggatgtttccaaaacatgtttttctactttatctct tacagGTTTTGGAATCATTTTCAAATAAAACTCCCCC TCACACCACCTGACTGGAAGTCCTTCCTGATGTGCTC TGGGTAGAGAGGACCTGAGCTGTCCCAGgtaaagcat cctgcaggtctgggagacactcttattctccagccca tcacactgtgtttggcatcagaattaagcaggcacta tgcctatcagaaaacctgacttttgggggaatgaaag aagctaacattacaagaatgtctgtgtttaaaaataa gtcaataagggagttcccatcgtggctcagtggtaac gaaccctactagtatccattgaggacacaggttcaat atctggcctcactcagtcggctaaggatccagtgatg ccgtgagctgcagtgtaggccacagacgtggctcaga tctggtgctgctgtggctatggtgtaggccggccccc tgtaactccaattcgacccctaggctgggaacctaaa aagaccccaaaaaagtcgctatgaatagtgaatacat ccagcccaaagtccacagactctttggtctggttgtg gcaaacatacagccagttaacaaacaagacaaaaatt atcctaggtggtcagtgggggttcagagctgaatcct gaacactggaaggaaaacagcaaccaaatccaaatac tgtatggttttgcttatatgtagaatctaaattcaaa gcaaatgagcaaaccaattgaaacagttatggaagac aagcaggtggttgtcaggggggagataaggggaggca ggaaagacctgggcgagggagattaagaggtaccaac tttcagttgcaaaacaaatgagtcaccagtatgaaat gtgcaatgtgggaaatacaggccataactttataatc tcttttttttttttgtcttttttgccttttctaaggc tgctcccgtggcatatggaggttcccaggctaggagt ccaaacagagctgtagctgccagcctacaccagagcc acagcaacacgggaaccttaacccgctgagcaaggcc agggatcgaacccgagtcctcacagatgccagtaggg ttcattaaccactgagccacgacaggaattccagggt ctgttgtgttcttaaaacacttccaggagagtgagtg gtatgtcataagtaaacaataaatgttaaccacaaca agcttatgaaataaacaggaaagccatatgacctaca atcagtcattgggagaatccacaaaaggttgagcaga ggatcaattccagctcacactccagttttagattctc ccctgccttaaagcatcacagactacataatctgagc tgaagaataaaaattaaaactcaccccagtgcaaaac agaaatgaaaaagtattaaaacgaggttcatactgtt gttcattagcaatatcttttattcacagGGGTGCCCA ACAACATGAAAAAATCAAGAATTTATTGCTGCTACGT CAAAGCTTATACCAGAGATTATGCCTTATAGACATTA GCAATGGATAATTATATGTTGCACTTGTGAAATGTGC ACATATCCTGTTTATGAATCACCACATAGCCAGATTA TCAATATTTTACTTATTTCGTAAAAAATCCACAATTT TCCATAACAGAATCAACGTGTGCAATAGGAACAAGAT TGCTATGGAAAACGAGGGTAACAGGAGGAGATATTAA TCCAAGCATAGAAGAAATAGACAAATGAGGGGCCATA AGGGGAATATAGGG

TABLE 13 SEQ ID NO. 49 TCTAATGCCTTGTGGAAGCAAATGAGCCACAGAAGCT SEQ ID NO 49 GAAGGAAAAACCACCATTCTTTCTTAATACCTGGAGA GAGGCAACGACAGACTATGAGCAG gcaagtgagagggggctttagctgtcagggaaggcgg agataaacccttgatgggtaggatggccattgaaagg aggggagaaatttgccccagcaggtagccaccaagct tggggacttggagggagggctttcaaacgtattttca taaaaaagacctgtggagctgtcaatgctcagggatt ctctcttaaaatctaacagtattaatctgctaaaaca tttgccttttcatag CATCGAACAAACGACGGAGATCCTGTGTGCCTCTCAC CTGCCGAAGCTGCCAATCTCAAGGAAGGAATCAATTT TGTTCGAAATAAGAGCACTGGCAAGGATTACATCTTA TTTAAGAATAAGAGCCGCCTGAAGGCATGTAAGAACA TGTGCAAGCACCAAGGAGGCCTCTTCATTAAAGACAT TGAGGATCTAAATGGAAG gtactgagaatcctttgctttctccctggcgatcctt tctcccaattaggtttggcaggaaatgtgctcattga gaaattttaaatgatccaatcaacatgctatttcccc cagcacatgcctaactttttcttaagctcctttacgg cagctctctgattttgatttatgaccttgacttaatt tcccatcctctctgaagaactattgtttaaaatgtat tcctagttgataaacagtgaaacttctaaggcacatg tgtgtgtgtgtgtgtgtgtgtgtgtgtttaccagctt ttatattcaaagactcaagcctcttttggatttcctt tcctgctctctcagaagtgtgtgtgtgaggtgagtgc ttgtccaaacactgccctagaacagagagactttccc tgatgaaaacccgaaaaatggcagagctctagctgca cctggcctcaacagcggctcttctgatcatttcttgg aagaacgagtgctggtaccccttttccccagcccctt gattaaacctgcatatcgcttgcctccccatctcagg agcaattctaggagggagggtgggctttcttttcagg attgacaaagctacccagcttgcaaaccagggggatc tggggggggggtttgcacctgatgctcccccactgat aatgaatgagggattgaccccatcttttcaagctttg cttcagcctaacttgactctcgtagtgtttcagccgt ttccatattaggcttgtcttccaccgtgtcgtgtcgt caatcttatttctcaggtcatctgtgggcagtttagt gcgaatggactcagaggtaactggtagctgtccaaga gctccctgctctaactgtatagaagatcaccacccaa gtctggaatcttcttacactggcccacagacttgcat cactgcatacttagcttcagggcccagctcccaggtt aagtgctgtcatacctgtagcttgcttggctctgcag atagggttgctagattaggcaaatagagggtgcccag tcaaatttgcatttcagataaacaacgaatatatttt tagttagatatgtttcaggcactgcatgggacatact tttggtaggcagcctactctggaagaacctcttggtt gtttgctgacagactgcttttgagtcccttgcatctt ctgggtggtttcaagttagggagacctcagccatagg ttgttctgtcaccaagaagcttctgcaagcacgtgca ggccttgaggtcttccgacttgtggcccggggactct gctttttctctgtccttttttctccttagtgggccat gtcctgtggtgttgtcttagccagttgtttaagggag tgttgcagctttatgattaagagcatggtctttcctt gcaaactgcttggtttagaagcctggctccaccactt agcggctctgtgacctcggacacatttcttagccttt ctgggcctcgctcttcttcctcataaagtgaaaatga aagtagacaaagccttctctgtctggctactgagagg atggagtgatttcatacacataaagcacttaaaataa tgtctggcatatgatacatgctcaataaatgtcactt acatttgctattattattactctgccatgatcttgtg tagcttaagaacagaggtctttacaggaattcaggct gttcttgaatctggcttgctcagcttaatatggtaat tgctttgccacagactggtcttcctctccttcaccca aagccttagggggtgaacgatcccagtttcaacctat tctgttggcaggctaacatggagatggcaccatctta gctctgctgcaggtggggagccagattcacccagctt tgctcccagatacagctccccaagcatttatatgctg aaactccatcccaagagcagtctacatggtacactcc cccatccatctctccaaatttggctgcttctacttag gctctctgtgcagcaattcacctgaaatatctcttcc acgatacagtcaagggcagtgacctacctgttccacc ttcccttcctcagccatttttcttctttgtacataat caagatcaggaactctcataagctgtggtcctcattt tgtcaatctaatttcacagcctcttggcacatgaagc tgtcctctctctcctttctgcctactgcccatgagca gttgtgacactgccacatttctcctttaacgacccag cctgctgaatagctgcatttggaatgttttcaatttt tgttaatttatttatttcatctttttttttttttttt tttttttttttttttagggccgcacccatgggatatg gaggttcccaggctagggatccaatgggagctgtagc tgctggcctacaccacagccacagcaatgcacaattc gagccaatctttgacctacaccagagctcacggcaac actggattcttaacccactgattgaggccagggatca aactctcgtcctcatagatacgagtcagattcgttaa cctctgagccatgatagttgttagttactcattgatg agaaaggaagtgtcacaaaatatcctccataagtcga agtttgaatatgttttctgccttgttactagaaaaga gcattaaaaattcttgattggaatgaagcttggaaaa aatcagcatagtttactgatatataagtgaaaataga ccttgttagtttaaaccatctgatatttctggtggaa gacatatttgtctgtaaaaaaaaaaaatcttgaacct gtttaaaaaaaaaacttgactggaaacactaccaaaa tatgggagttcctactgggacacagcagaaatgaatc taactagtatccatgaggacacaggtttgatgcctgg cctcgctaagtgggttaaggatatggtgttgctgcag ctccaattcaacccctatcctgggaacccccatatgc caccctaaaaagcaaaaagaaaggtgctgccctaaaa agcaaaaagaaagaaagaaagacagccagacagacta ccaaatatggagaggaaatggaacttttaggccctat ctccaactatcacatccctatcaccgtctggtaagaa atggaaaaaatattactaagcctcctttgttgctaca attaatctgattctcattctgaagcagtgttgccaga gttaacaaataaaaatgcaaagctgggtagttaaatt tgaattacagataaacaaattttcagtatatgttcaa tatcgtgtaagacgttttaaaataattttttatttat ctgaaatttatatttttcctgtattttatctggcaac catgatcagaaatctttaaacaatcaggaagtctttt ttcttagacaaatgaaaatttgagttgatcttaggtt tagtacactatactaggggccaagggttatagtgtga ctattaaatcacagataatctttattactacattatt tccttatactggccccacttggatcttacccagctta gctttgtatgagagtcatccttaaagatgactttatt ctttaaaaaaaaaaacaaattttaagggctgcaccca tagcatatagaagttcctaggctagcggtcaaattag agctgcagctgccagcctatgccacagccacagcaat gccagatctgagctgcatctgtgacctacactgcagc ttgcagcaatgctggatccttaacccattgaacaatg ccagggattgaacacacatcctcatggatactgctca ggttcctaacctgctgagccacagttggaactccaaa gcagactttattctgatggctctgctgatctctaaca cgttattttgtgccatggtgtttatcttcactttact caagtcagggaaacacgaagagtctcatacaggataa acccaaggagaaatgtgcaaagtcacatacaaatcaa actgacaaaaatcaaatacaaggaaaaaatatcttca ctttcaaaatcacctactgatgatgagtttatatttc cttggatatttgaatattagctatttttttcctttca tgagttttgtgttcaaccaactacagtcgtttacttt gatcacagaataatgcatttaagccttaaatagatta atatttattttcaccatttcataaacctaagtacaat ttccatccag GTCTGTTAAATGCACAAAACACAACTGGAAGTTAGAT GTAAGCAGCATGAAGTATATCAATCCTCCTGGAAGCT TCTGTCAAGACGAACTGG gtaaataccatcaatactgatcaatgttttctgctgt tactgtcattggggtccctcttgtcaacttgtttcca atctcattagaagccttggatgcattctgattttaaa ctgaggtattttaaaagtaaccatcactgaaaattct aggcaagttttctctaaaaaatcccttcattcattca tttgttcagtaagtatttgatgagaccttaccatgtg taaacattgcactaggtattaagaaatacaaagatgg ataagatagagtcggcgtaaatgagatgatataatga gacgttataatgaaactcacaattccagttgggaaat aaagtccttcaaattccatgactctttctggcacacg ttagaggctacagcttctgtgtgattctcatgctggc tccacttccactttttccttcttcctactcaagaaag cctatagaaatatgagtaagaagggcttaatcatagg aataaatttgtctctgttctaagtgattaaaaatgtc tttatcagtataaaaagttacttgggaagattcttaa aactgcttttacacactgttctagaatgactgttata taaataaaaaagtagatttgatctaacacaattaaat gacctttggaaatattgactaattctcaccttgcccc tcaaagggatgcctgaaccatttccttcttttgccag aaagcccccaccctttgtctgttgacctagcctagga aatcttcagatcacgttgttagcacgaactggttaca tgtgctgtacaaatactatttaattcatctgattaaa aaaaaagagataagaagcaaaagtttgactatcttaa actgtttgcgtaggtgagaggacaattgaccatctac tttatgagtatgtaacccagaaacttaaagctcctta agggagctaagtcttttggataagacctatagtgaga ccttttagcaaaatggttaagactgaatggagctcac tagcgtgggttcatatcctgatgctcaaacacgcaat taaatgactttaggtgggttagtctctgttccttagt ttcctcaatgggagataatattggtagtagcgatttt actgggttgttgaaagaacatctgttaaatgttcaga acgtgttacgacagagtacagagtaatgatttgcttg tatatgtatgactcaaatagtctgccatatgccttgt gactgggtcctgtggagcaggaaggagggatttccca cccagcagaaagttgggtaaactggaaaatagactga ggccaggaaatgatgcaaagcgttgatgttcactgcc acggcaggtgaagggcagggccagagttgtcagtagg gtcaggggaggactggaaataaccaagacccactgca cttttcagcctttgctccagtaaggtaatgttgtgag agtagaaaattttgttaacagaacccacttttcagta cagtgctaccaaactgtagtgatttcataccacatcc caagaaagaaaaagatggctcaatcccatgtgagctg agattatttggttttattgttaaataaatagcattgt gtggtcatcattaaaaaaggtagatgttaggaaagta gaaggaagaagactctcacctacattttcatcactgt tttggtatctgccagttgtcaccttggtccccttccc cgcctctcccctgcctcctcttcctccttctcctttt tttggaatacaattcaggtaccataaaatttaccctt ttagagtgtttgactcaatggtttttagtattttcac atgttgtgctattactatcactatataattccaggtc attcacatcaccccccaaagaaaccttctaactatta gcagtccattcccttcttccctcagcccctggcaacc actaatctacttactgtctccatggatgttcctatat tgaatcaagctagcataaaccccacttgctcatggtc ataattcttttttatagtgctaaattacatttgctaa tattcaattaaggatttctatgtccatattcataagg aatattggtgtgtagttttctctttgtgtgatatctt tgtctggttgggggatcagagtaataattactgctct catagaatgaattgagaagtgttccctccttttctat ttattggaagagtttgtgaagtatattggtattgatt cttctttaaacatttggtcagattcaccagtgaagcc atctgggccatggctaatctttgtgaaaagttttttg attactaattaaatctctttaatttgttatgggtctg ctcctcagacgttctagttcttcttgagtcagttttg ttcatttgtttcttcctaggactttctccctttcatt tggattatttagattgatagtaatatcccccttttaa ttcctggctgtagtaatttgggtcttttctctttttt cttggtcagtttagctaaaggtttgtaattgtattaa tcttttcaaataactaacttttttgttttgtttgttt tttgttttttgttttttgttttttgtttttttttgct ttttaaggctgcacctgaggcatatggaagttctcag gctagaggtctaatcggagctacagctgctggcctat accacaaccatagcaatgccagattcaagctgcatct gcgacctacaccacaactcggccagggatcacacccg caacctcatggttcctagtcggatttgttaaccactg tgccacgacgggaactcccgcccattttttttaacac ctcatactttaacataaagatgggcttcacatggact gatagctcaaatgaggaaggtaagactatgaaagtaa tggaagaaatgtagactatttttgtgacctagagatt actgatacttcttgacttttcaaacaatacttcaaaa gtacagcccaaagggaaaaaagaaagaaaaaagaaac acacatatacacaaacctagtgaataagatatcatcg atacactacagatttctatgaactggaagaccccatg gacaaagttaaagaacatatgatagtttgagtgatta ttttgcaatatttacaaccaatgagggaatattatcc agcttataggaggaagtaatgcaaatcgacaagaaaa agataggaaacccaatataaaaattaagaaaatacaa aaattaagaaaggatatgaactagcattttacaaaag aaaaatctccaaaagtcaatcagcacatgaaaatatg ctcaaacctattaattattagaaaactacagactgaa gcaatgaggtgctttactttacatctttttgactgat aaaaagttagaaacaaaggtgatatcaaatgtcaggg ataaaaggatatagaaatcgtcatgcctgtggtggga gtatggccggtgcagtcatgtgggaaggtaatctgac agtggttaggcagagcaggtttatgaatacactgtgg cccatcaatcccacgcctgtttatgtaccaaagaaat cctgttgtggcagaatctatgggtccacccctgggag catgaattaataaaatgtggcaccagggtgtgtgaaa ctccagctagagatgagatgtccacatggcaacatga atgcatcttagaaacatagatttgagtgaaaaagagt aagaaacagccgggaaacccaataccatttataaaaa ttaaagatgcacacatacaatgtagtaaatattttgc atgaactttcaaatggttgcctacagggggggagagt aaagaagagtagaaaacaaagataaagggagtaagta agtagctctgcctggactgaatataatgtgtcatgaa ctgagaaatatggttaacataatcctctaacttgagg tcctaaatgaatgaatgagtccactattcatttaccc attctttaatgtgtattgcattataatccattttttt agaaccaacgaattttgttcccataactactaatcag cctgccttttctccctcattcccttatcagctcaggg gcattcctagtttttcaaacgttcctcatttgaacca aaaatagcatcattgtttaaattatacttgttttcaa atacgatgcttatatattccaagtgtgtttgcccatt ttcttaggtggtagaaatttttcattctacttttcta tctactcagattttcccgttggaattatttccattgc tattaaacttagaagtcccccctgtgatatgccattt ttttcatactttttaagcacttggttgcttttctttg tgtctttaagcacctagaatacttataaccattgcac agcactgtgtatcaggcagcccttcctcttccactaa tttatggtccttctcttagactatattaaactgttat ttaattaggatcctctcttcgtccttatgatttaatt attatagttttctaatatgtttttattataattcctc ttcattattcctccctattaaaaattttaatgaattc catttgtttgttcttctagttaaatattaagtcataa tccaaataacttagatgtcattagtttatgtggtcaa agtaaggataccacatctttatagatgcaggcagttg gcagatgtcatgattttcttcagtgcataaatgcaat ttatctttgagcaaggggcataaaaacttttatggta ttggctttgaaataatagttaagaactgcagactcag tttttcctgcttttcttgaaaaagaacacttctaaag aaggaaaatccttaagcatggatatcgatgtaatttt ctgaaagtctcctgtaattccttgggatttttgttgt tgtttgttggtcggtttttttgggtttttgtttgttt gttttgttttgttttgttttgcttttagggctgcacc tgtggcatatggaagttcccaggctaggggtccaact ggagctacagctgccagcctactccacagccacagca acatgggatcctagctgcatctgtgacctaaccacag ctcttggtaatgccagattgttaacccactgagcaat gccagagatcgaatctgcctcctcatggacactagtc agattagtttctgctgagccacaatgggaattcccaa ttccttgtatttttgaactggttatgtgctagcatat aattttgtttcttgaatctttgtgggttttttttttt ttttttttttgtctcttgtctttttaaggctgcaccc acagcatatggaggttcccaggctagaggtcaaattg gagctacagctgccagcctacacaacaactgcagcaa agtggggcccaacttatatgacagttcgtggcaatgc cggattcctaacccactgagcagggccagggatcgaa cctgagtttccagtcagtttcgttaaccactgagcca tgatagtaactcctgtttgttcagtcttgaacctcct ttttaattctttattccttgagggtgaaataattgcc ataataatactatcatttattacatgccttctctgtg ctaggcatagtgacactttaggatttattatatcact taatccctacaacaactctgcaaagtatgtatcataa tcctatttgacagatcaggaaattgcagcccaggatg cagataatatgcatccatcacaagtgactagatatag tccctctgctattcagcagggtctcattgcctttcca ttccaaatgcaatagtttgcatctattgtatatgtgt tttggggtttttttgtctttttttttttttttgtctt ttctggggcctcacccttggcataggtaggttcccag gctaggggtcaaattgaagctgcagctgccagcctac accacagccacagcaactcgggatctgagcctcatct gcaacctacaccaaagctcacggcaacaccggatcct taacccactgagtgaggccagagatcaaaccggcaac ctcatggttcctagtcggattcattaaccactgagcc acgatgggaactccctaaatgcaatagtttgctctat taaccccaaactcccagtccatcccactccctcctcc tccctcttggcaaccacaagtctgttctccatgtcca tgattttcttttctggggaaagtttcatttgtgccat ttttcattttacgggtaatttttacttcagtttcttc cactagcagttgtcttaaagtgagtataattaatatt catttggaaaatgtaagcaaaacattttttaaagggc catgcccacagcatatgaaagtttctgggccaggggt tgaatccaggctccaagttgcagctgtgccctacact gcagctgggcaatgctggatcctttaacccactgtgc ccggctagggatcaaacctgcatttccacagctaccc gagccattgcagttggattcttaacccactgcactac agtgggaactcccacaaaacattttttaatgtccttt gaataaagtaggaaagtgctcgtctttgagggcaggg cggcaatgccatttccacaaggtgctttggcttggga cctcatctgctgtcatttagtaatgaataaaattgct gacagtaataggattaactgtgtgtggagatagccag ggttagagataaaaacactggagaagtcaaataagtt gctcgaggtcctctagctaataagctattaagtggga gagtgagggctagaaacaggccatctgtctcccaagc acatgtccattagtggtttgctgatagccttccagaa caacagagaggactctcaaacatggtcttgcctccct ccaattgatcccctccatgtgcctcacagcgggtctt tctaaaattaagttctgattttaattctcccttgcta tagcacttaggtatggctttcagccgtgcaataaaaa gcaggcaagagtggctcaatcatataggaggttgttt ttcttagatcccaagcaggtaatcctgggcattatgg ttgttctgcgtttatcaaggagccaaattctctatca cctcctgttctatcctcctcagtatctggctctattc ttcagcatctcaagatggcttgtgctcctccaagcat ggcagtcaaattccacacaagagggggaaaatgaagg gcagacagtgctggtctcctgagctgtccctctttgt cggggaaataaatgtattccttcaagtcccgtgagac ttctgaagtagacgtctgcttacgtctcacccaccag aactatgtaaactgcacatagtgctaggtctacatag ccactcataactgccagggggtgggaaatctttaaat aggtgtaccaccacacaattaggatgctaatagtaag ggagaaggagagaataggttttgcgcaagccaccagc atgcctgccacaattgcttaaaattcttcattgaccc ctcattgccacaggatgaaatccaaacgccttcttag ttgggaatctgacctacctgtctctcccacctggttc agacaccattctccttggtcataaaattccagtcatt tgtgaacatccagctcccccatgcctccatgcctttg cacatgctgttcttttatcttttatgttgtcctttta tcttttatccaaaagagatatcccatcatcacatctc ttcagcccccaaatactttgtctttcaagttcagctg gaggattacctcctatttgaaatcagctttgtctctt acaaccaaacaaggttttccttccgagacactcccac agcaccttgaactcatctctatcaatcattcatttga tgtaatgaagttgttggtggtatgcctgtgtctctga cacatctgcgatctcatgagttccttaagtggaatgt gaatagcgggatgaacagtattggtcttcagccctca tctctgcagatgttgcttgacccaaatgagcgttgcc ttttattttgattttgctttgatttgtctactccatg tacttgagccatgcatttctgtcttagcgatgctttt taaaagtcattttttggttgattatccagatttgtcc acctttgcttctag TTGTAGAAAAGGATGAAGAAAATGGAGTTTTGCTTCT AGAACTAAATCCTCCTAACCCGTGGGATTCAGAACCC AGATCTCCTGAAGATTTGGCATTTGGGGAAGTGCAG gtaaggaaatgttaaattgcaatattcttaaaaacac aaataaagctaacatatcaatttatatatatatatat atatatatttttttttttttttacatcttatattacc ttgagtattcttggaagtggctagttaggacatataa taaagttattctgaagtctttttttttctttttccat ggtgagcagtggcttgatgtggatctcagctcccaga cgaggcactgaacctgagccgcagtggtgaaagcacc aagttctagccactagaccaccagggaactccctatt ctaaattcttgagcacattatttaggaacctcaggaa cttggcaggattacaggaaatatatctagatttaaaa aaaaatcttttaacagaggtcccaaaggagagtcatg cacagctatgggaggaagttcagaaactgcccttgct accagatcactgtcagataaaatggccagctacatgt ttctgcacattgccctaagatctttacaaacttttct gtgcatttttccacttttaaaagaaaatttcggggtt cctgttgttgctcagtggttaacgaacccaactagta tccatggggacaggggttcgagccctggcctcactca gtgggttaagaatctggcattgctgtggctgtggcgt aggctggcggctacagctcagattggacccctagcct gagaacctccatatgccgcaggtatggccctaaaaaa aaaaaaaaagagagagagagaatttcctccagaaaaa acactttggtagtttgggagaagtaaacaaccaaaaa ttaatttttctggagtattcgggaagcttgtaaaaat gggctcttacttttttgaggagacaaatgggaaccta cccagaagaggcacaatcacctgcatttgatttcttg acctctccctaccttctttgctggctttccacatttg gatttctgtgaccttatctctgctccttggtgttttc atttttcctgtggacgtgccagactatgggaagggag taaggcgttgatttagaatcctgtagtctctgcctgt ctctagtcattgttttcacccttctcaaaggaccttg acatcctgagtgagtccgcaagtaatttaggggagaa gccttagaagccagtgcagccaggctacatgactgtg tccacccactggaaccagtcatttttatacctattca cagcccccctaccatttaaatccccagaggtctgcca taacatctgtaactccctttcctggtaaattgtgttc taaaagactggtaacaaaagatattctgtggtacaga gcataattaaatacctgggagctgatttgagtggggt aaatcaactggtttgacccctaaaacccaccatgagc atttctgttctaataaagtaatgcccgtgctgggaat tgtgttctacggaaatgctcctgctgtgtctttcttg agtcctgtgtcattgaacatgcttaggagcaaaggtc ccccatgtggcttgtctgctaaccagcccagttcctt gttctggctggtaatgatccgatcatctgaatctcac tgtcttccaacag ATCACGTACCTTACTCACGCCTGCATGGACCTCAAGC TGGGGGACAAGAGAATGGTGTTCGACCCTTGGTTAAT CGGTCCTGCTTTTGCGCGAGGATGGTGGTTACTACAC GAGCCTCCATCTGATTGGCTGGAGAGGCTGAGCCGCG CAGACTTAATTTACATCAGTCACATGCACTCAGACCA CCTGAG

SEQ ID NO. 49 represents contiguous genomic sequences containing Intronic sequence 5′ to Exon 4, Exon 4, Intron 4, Exon 5, Intron 5, Exon 6, Intron 6, Exon 7, Intron 7 and Exon 8 (Table 13). Further, nucleotide sequences that contain at least 1750, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, or 20000 contiguous nucleotides of SEQ ID NO. 49 are provided, as well as nucleotide sequences at least 80, 85, 90, 95, 98, or 99% homologous to SEQ ID NO. 49.

TABLE 14 SEQ ID NO. 50 AGGAATGGAAAGCCCAATTCATTAAAACAGAAAGGAA SEQ ID NO. 50 GAAACTCCTGAACTACAAGGCTCGGCTGGTGAAGGAC CTACAACCCAGAATTTACTGCCCCTTTCCTGGGTATT TCGTGGAATCCCACCCAGCAGACAAGTATGGCTGGAT ATTTTATATAACGTGTTTACGCATAAGTTAATATATG CTGAATGAGTGATTTAGCTGTGAAACAACATGAAATG AGAAAGAATGATTAGTAGGGGTCTGGAGCTTATTTTA ACAAGCAGCCTGAAAACAGAGAGTATGAATAAAAAAA ATTAAATAC gtatggctggatattttatataacgtgtttacgcata agttaatatatgctgaatgagtgatttagctgtgaaa caacatgaaatgagaaagaatgattagtaggggtctg gagcttattttaacaagcagcctgaaaacagagagta tgaataaaaaaaattaaatacaagagtgtgctattac caattatgtataatagtcttgtacatctaacttcaat tccaatcactatatgcttatactaaaaaacgaagtat agagtcaaccttctttgactaacagctcttccctagt cagggacattagctcaagtatagtctttatttttcct ggggtaagaaaagaaggattgggaagtaggaatgcaa agaaataaaaaataattctgtcattgttcaaataaga atgtcatctgaaaataaactgccttacatgggaatgc tcttatttgtcag GTATATTAAGGAAACAAACATCAAAAATGACCCAAAT GAACTCAACAATCTTATCAAGAAGAATTCTGAGGTGG TAACCTGGACCCCAAGACCTGGAGCCACTCTTGATCT GGGTAGGATGCTAAAGGACCCAACAGACAG gtttgacttgaatatttacagggaacaaaaatgattt ctgaattttttcatgtttatgagaaaataaagggcat acctatggcctcttggcaggtccctgtttgtaggaat attaagtttttcttgactagcatcctgagcttgtcat gcattaagatctacacaccaccctttaaagtgggagt cttactgtataaaataaactattaaataagtatcttt caactctggggtggggggggagactgagttttttcac agtcctatataataattttcttatcctataaaataat taggagttcccgtagtggctcagcaatagcaaacccg actagtatcgatgaggatgcgggttcgattcctggcc cccctcagtgggttaaggatctggcattgccgtgagc tgtggtgtaggtggcagacacggctcagatcccacgt tactgtggctgtggcataggccagcagctccagctct gattagacccttagcctgggaacttccatatgctgtg ggtgtggccttgaaaaaaaataaataaataagataat tactcaaatgttttccttgtctcagaaccttacttca ggataaagagtgagaaagttttttttatgaagggcca ttattacagctcaaaaataagttgtcttcagcaagta gaaagcaataagcctgagagttagtgttcctatcagt gtaaatattacctcctcgccaatccccagacagtcca tttgaacaattaacggtgccctgggagtacagttcag aaacattaatgtggatgttccagacctgtatttttat aagtaccttgagccggatggaaccatcattcctcacc attatttagaagtggactgtgactctgttggagatca gggcacacggttaccaaaagcacacccttctcctggc cttacctttgcaaagctggggtctgggacacagtcag ctgattatacccttttactaacttcccacagctcaaa tctggtcaattctccttcacaaatctcttaaaaatcc atcactcacctccagcctcttctgctgtggccttgat tcagcctctcacaatttttttttaaccagaattctgg cagtggcccctgacttgcctctgtgctcccagccccg ctgtcctctgatccatcctccatgccagcctttttca atctgctggtcacgattcattgatgggttaggaaatc aatggcatcacaactagcatttagaaaaaggaaatag gcgttcccgccgtggcacagcagaaataaatccgact aggaaccataaggttgcgggttcaacccctggccttg ttcagtgggttaaggatccggcattgccgtgggctgt tttgtaagtcacagacatggctctgatccggcattgc tgtggctctggcgtaggcctgcagcatcagctccaat tagacccctatcctgggagcctccatatgctgcaagt gcagccctaaaaaaaataaaaaaataaaaaaaaataa ataaaagaagtagacaaattgtatagaacaaccctga gtatgttgcctgagcacatataacaagggtaagtatt atttcaggaaactctggtttcacagatactcttggca tatggacccctagagtcctgatgtaaaatatattctt cctgggatcttaggcaagaagtttgaaagctccaact ctgcactgctgccaaagaaatgatttttaagtgcaaa actcttcccgttcccttccctgtataaaattccatag gatctctccagtgcctctaggataaaggcagttttca ttctctagttcaaggtgagagaagattttaattattt cacgttttagtggggaattcaagagtctggcacctga catttgctgaactctctccattatccctctctagttc cccagacgcatcctatggtagaaattcgcaaactaga gtgagcgtcagagtaacccaaggaaactgggtaaatg cagctccctgggctctaccccctgagattctgattca gtagatctgaagcagagccctggaatatgcatatgca tcattgtgtcacaccaagcattctgggtaatgagagt tgatgttaggttctcagtagtaagacaagtatagaga ttccgggggactgagtgctcagctctgccttggggag gagggagagggctaaagagaacaggagatggggacag ggaatgctcaacctccaatcttaggcatttgagctat gtcttaggggtcaggaggaggttaccaatatagtgat taagagattgaggttccagtcagagggatatgctgga gaaggggggtgaaaataatgtcataggtttggtgagt gcagatactttgagttttttaatatttttattgaaat atagttgatttacaatgctcttagtgagtacaattac tttgaataagtgcatagatgtatgccattcttccaga aatgatttattgagctcctttgggcatcatgctaagt acaggggaaacagctgtgaagaggtccttcccttatg aagtcattcatccccttcagtaaatgaaggtaaagga aaaggatgagacagggacgccgtgttggaccagggtc agaaaggccttataagaccttgcctggagggcaagga acttgcctgtgagtaaggagagcttgagaaagcgata aagcaaagaaggaacattactgcattgtgttttagaa aaaccatgtcctggggaagaactcctagagtcagggg ggccagttgggagactgtgcttttttccaggaggaga taagtgaggctgctggctgagatggagcaaggattta gagaagcagatatgagattcatttagaagttagacat tttaggatctgacacataatttatcaccaaaaccagt gcatctctggctttgggccaccagttttggagaagtg gaatgtagggacctaccattacctgccaatctttact acacagatgcctatttccctcctcatatttcctttct ccagatcacgtcctattctattgccaggactcaagat tccaccttgcatgcagtgatccatcttcacactggat ggacagctctagggatgtcagagcacactcttgtcca tactgctgactgggtctcctgtcagcccatctgtcta tcagctgtggtattattagtataataagagggctgta tatgagagacacaaaattctaggtgtagctcaaagat aggctagagttattcctatgtacaacaaatatttatg ggaccccttctgactgtcatggttgctgctttcatca tacttgtagtctaatggaggtgggggcagggcaggaa taagcggatgtccacaaaatcagtaagaccacttata ttcaacattttcataatttagttatttgagcccaaag ggtccacatccgtggtattccaacttttttttccccg gacatggatctttatctttttttttttttcttttttg cggccagacctgcggcatatggaagttcccaggccag gggttgaatgggagttgcagctgcctggtctacacca cagccacagcaaggtgggatctgagctgcatctgtga catacaccgcagctgaggtaacaccagattctgaacc cactgaatgaggccagggatggaacccgtctccttat gaacactatgtcatgttcttcaccctctgagccacaa cgggaactccagacttcgtctttaaatgtattctgac ttggagagctatcacactaagcaattaacaggagctg acctggtttaggctggggtggggccctactcctcaat gttccctgaggcacatctgtgggacccctgggcatca tctatctgagcagccttagagctgctcatccagttga ctgttgatgtagaagtgcaaacttctgccttccttat ttgttgctttcttttttcattgttctctcccctttgt gtctttaag CAAGGGCATCGTAGAGCCTCCAGAAGGGACTAAGATT TACAAGGATTCCTGGGATTTTGGCCCATATTTGAATA TCTTGAATGCTGCTATAGGAGATGAAATATTTCGTCA CTCATCCTGGATAAAAGAATACTTCACTTGGGCTGGA TTTAAGGATTATAACCTGGTGGTCAGG gtatgctatgaagttattatttgtttttgttttcttg tattacagagctatatgaaaacctcttagtattccag ttggtttctcaaaagcattcattgagccttactgact gtcagacggagggcgtattggactatgtgctgaaaca atcctttgttgaaaatgtagggaatgttgaaaatgta gggaatgaaatgtagatccagctctgtttctcttttg gaggattctttttcctccatcaccgtgtcttggttct tgtttgttttgggtttttgtgggtgttgtattgtgtt gtgttggttatggcagtgacagctatttaaactgtga aacgggggagttcccgtcgtggcgcagtggttaacga atccgactgggaaccatgaggttgcgggttcggtccc tgcccttgctcagtgggttaacgatccggcgttgccg tgagctgtggtgtaggttgcagacacggctcggatcc cgcgttgctgtggctctagcgtaggccagcggctaca gctccgattggacccctagcctgggaacctccatatg ccgcaggagcggcccaaagaaatagcaaaaagacaaa ataaataaataaataaataagtaagtaaaataaactg tgaaacggggagttcccttcatggctcagcagttaac aaacccagctaggatccatgaggatgtaggttcgatc cctggccttgctcagtgggttaagaatccagcgttgc tgtgagctgtgatgtaggtcgcagatgcagcccagat cctgcattgctgtggctgtggcgtaggctggcagctg aagctccgattcaacccctagcctgggaacatccata tgctgcaggtgtggccttaagaggcaaaaaaataaaa aaataaaaaataaataaattgtgggacagacaggtgg ctccactgcagagctggtgtcctgtagcagcctggaa gcaggtaaggtaaggactgcagctgggtaaggactga attgcaccaactgggaagtaagcctagatctagaact taagttagccctgacatagacacacagagctcaccag ctaagtggttcagcttataagctggtcactgaaactg aggatgtccacaaaagcaaaataagtagcaacaggca gcgggatgcaagagaaagaggaggcctaaaatggtct gggaatccctgccatacctatattttatcctacttat atttagtgcctgaatgtgtgcctggagagcaaagttt agggaaagcatcgggaaatgcacagtattcataccct taggaacaaagatcagttacctccagggtaaagacta tttccaagtttaaatttcaacccctgaacattagtac tgggtaccaggcaacacttgccatcctcaaaatcaat gaatcctaaaattcaacctgggggtcagtgacagtct gtgacaaagtttttgctggtcagtaacgaaataagta tgagcaccatctgagtatggtcaccaagatgtcaact ctctttcctttggacgaattgtcaltattccaagatt aggtcctttctatttttgaggtgtgaaaacatctttc ctttcataaaataaaaggatagtaggtggaagaattt tttttgttttttggtctttttgctatttctttgggcc gcttctgcagcatatggaggttcccaggccaggggtc gaatcggagctttagccaccggcccacgccagagcca cagcaacacgggatccaagccgcatctgcagcctaca ccacagctcacggcaatgccggatcgttaacccactg agcaagggcagggaccgaacccgcaacctcatggttc ctagtcggattcgttaaccactgcgccacgacgggaa ctcctaatgatactcttttatatttagctactatgtg atgatgagaaacagtccacattttattattttttagc caatttgatatctcattactaagataatgataatttt ctctataaattttatttaagttagtgttatgaagtgg ttttgctagtgtagaaggctaggatttgaattcagtt caagaaagaagagagggagggagggagagggatgggt agagggatggggcagtgggagagagcaaagaggagag acagtttttgtattaattctgcttcattgctatcatt taagggcacttgggtcttgcacattctagaattttct aaggaccttgaccgccagattgatatgcttcttccct ttaccatgttgtcatttgaacag ATGATTGAGACAGATGAGGACTTCAGCCCTTTGCCTG GAGGATATGACTATTTGGTTGACTTTCTGGATTTATC CTTTCCAAAAGAAAGACCAAGCCGGGAACATCCATAT GAGGAA gtaagcaggaataccagtggaagtgcccctttcttcc ttccttcctaaataaacttttttattttggaacaact ttagagttacagaaaagttgcaaagatattatagaca gtagtgtttatatatatatataaatttttttttgctt tttatgaccacacctgtggcatatggaggttcccagt ctaggggttgaattggagctacagctgccagtctgtg ccataaccacagcaatgcaggatctgggccacgtctg tgacctacaccaaagctcacagctggattcttaaccc actgagcaaggccagggattgaacctgcatcctcgtg gttcctagttggattcgtttccgctttgccgcaatgg gaactccaaattattgttaatatcttactttactggg gtacatttgttacaaccaatactctgatactgaaaca ttactgttaactccgtacttgcttctttttgagtcat ttgcaaagactggcttcttgacctgcttccttccaaa cagctggcctgcctatgctgttctcagacctgcaagc actgatctctgccccccttgccttctctccagtggtg tctccttccccaaacaaacccagtgtggctctggaaa gggagttaagtcaacataaaccaacacatattttgtt gagctccaattttgagcaaatccctcaccacggcaga caggcatgatgttaagaactagggctttggacacaag gtcaagaccaagaagggttcctcacccctactgattc agataaccaataatgaggctttgaatccctgtccaaa ggttgttttttttcccttctattgagcttcttgccac cttatcagttttttttatgacagtcaaatgacatgat atatgtgagcatacatggtaatttttaattctatata aatgaatcactaaataaattaggaggatatatagtcc acctttaagcgtattacacgtgtcacatgaatgtgtg gcgacttaattgtagaggtttaaatgtagcttcctat aatagatgtgttcctaaactacattttaatcattgga cttgtatttttatgttagcacttgctgttgaagaaaa gcctatgccaaaagttcagtgaaaccaataatccact gccagctttctgagttaaaaaaaatccctgggttttc acacacaggaacaccctgtgtgaaacactcatttaga gcaaaatgcatctgataaggagttcctgttgtgcctc aactggttaaggacctgacattctccatgagaatgtg agtttgatccccggccccactcgatgggttaaggatc tggtgttgccacaaactgcagctccgattcatctcct agcctagaaacttccacagcccagaatatgccacaga attcggctgtttaaaaaaaaaaagaaaaaaaaaagaa tcataaatgtgttggtttgttcaccaaatacatgata acttgctcttgccaagctcagcttcataaatattaag tcatttaatacagcagccaccttatgaacagatatta ctatacttcccatttacagataaggaaaatgccatat ttaaccaagagattaaataactttcccgaggtcttat agcaagtaaatcatggtgcaggggtttgaccacacgc agtctatctccagagtctgtgtatttagccactgttt tactttcaaatttaaatttataaaacttctaaattat ctgttaaccataatctttggaatttttaaaaccacga gttcctataaaatgtttcattgaaagtaagtcacttt tccatagcttttgataatacatctgtaggataaagta agccacagctctcttgcagacttggtacaccctgggg caaagcatcatgcctgtcacgtacatggtggtcctta ctttgactctcagtgcttttattgcccaggaattttg tgagatttctagttgttgaggtttgtttaaagaggtt atgccggtacttggaagagctcttttcttgctacctg gagccttctcatatttcctttttgaggagggacatga attgcctttcaaactcataaatatattttctagtaca caagtctccatcttccttagacgcatggctcctggag ttctccatcctcctgctccactttgggtgggctcctc tctgggtctgccaccaatctgccacccagagacatcc ttgacccacttccagaccccaccatggcttcactttc ttcgctttcctcctttgtggaaccttctgcttaagaa tctgaggaagaaaatttgcacgtgagctaaactggag gtactttcctgcctggtcttgcacgatagcttggctg agcccatgatgctgggtggctgttactttccatggac acccgaaggcgttgctcctttggcttctagttgcatg cagtgttgcttatcccaggctgatctttcttccactg taggtgacttttaagaattaagggattaatctatatc tacaacaacaacaacaaagaccttttcaagctgaggt agggctttctgtatatgtttggagtggttatccagca gactttacttgaaggcaggggtcatatcctcaagtgc tcataaacggaccacagaaagatctcataattgggtg gagctgggtggggaccgtgtcatgtggccaggaaatg ccagatgggaagggagtggcccttactgagctccagc tgaactctgaattttctagaaaactcagaaatctgga tttttcatgtgtaatacccagatttatagatgtggaa agctaattctttttttttttaagggactataggcaat gaactaagatctaggttgtatttggacaaggggtcat cagtttaagctgtgtagttgagcgctcagctattggg ctgagggacccctaaatactgagacggggaggtcctt gctctggggcatcacaagtacactccctggtctcatt caaacacttttcctacaaaattgatcccatttcttca gtgcactgtctgaatgcatttggcccagagccgtgct gaggcatagggaaggggtccacggtttcatggcatcg ttttgtgctgtgtgtccctgctgtcgtccaggatacc tacctctcctcctcctgcatctgaatgtccccccaca gactctctgggattctacagcctctggcctgttcctc agacacctcttacctgccagctttccagattcacatt agttagtccaaatctactgccgtcagtgactcacttc atttcttcttctccgaggcagttcagcccggtacagt tgttttgtcaacacttcagttgagtctggaagatgtg catgggttatgcacgagagcggtccatcattttgagc tagaagtcctttctcagcccagagacaagtcctcatc tcctttacttcctgactcttcttcctctgcatccttc caagatatctctttctccagccaccacctaaatctct tcttttcccggggttccgtgctcaacccactcttctt cttaaatctgtggctgggtgaacgcatctgctggcac cacttctctgctaaagactccaaaatccataggtcct gcccggcctttgcccacctctctccaacactgtccag ctttagatgtagagctaatccccccagagatatcatt ccctggatgtctaagtcctttggtatctcactttcag cgtgttcaaaatcctcttacaactgttctttctcctt ttccatcttgattattggcaacatgccagcctttccc ctacccccagcagtgagccaagctagaaacaagggct taatcttcaatctttccttctccatccctaaacctaa tgagtctccaagcccttcccagtttacaccctaaatg ttgctcaaaacatcccctagttcttccacgtgctctc ctctatattgaaaggtcaagaaaggccatcttccctc cactgtgaggaaatagatcttgatactgcccctgagc tgggcagtcctcgacctgacaaactgtgcagtgtttc taaatctctactggcaaaatgagagtgcctttgacct gtgttgcgatctcagatcacagtggatgtaattgttt tataggaatggtgaacgaaaaagaagtaaatccctaa tgccaaactcctgatcattctatgtcatttaatagcc tgtcatttatgataaagtttcctctactggcattagc acaatacttctcaggaaaaaaaaatatgatgccagat actgaaaagctcctgggtaaacatgaacatgggtacc gataaaatggtgaagccagtccaatcttagagtgact tcccttcatgctacttcatgctctttttttttttttt ttttaagaaaaaccccttttttttttctcacaccagt cacagaggagaccgaggcttagcaaggttaaggtcac atgattagtaagtgctgggctgaaactcaaaaccatc tctgcttgtctcctaaccctgtgcacctctgactatt caacag ATCCTGTGTCAGGAGTTGGGATTCTTTGAAG gtaagggccttgaccaccgaattaaggtaatcttgct ctgtggcaggccttgttttcagtattttaagtacact ggctcaggtaatcctcacaacagccccaggaggaatg ttctattacctccactgtatagatgaggaacttgagg cacagaatggttgccaaggtcacacagctatattggg ggttcatacccagccatccaactctgtctgtactctc tgccactctgcacccccagctcctgatccacttcctg tttccatccctcgatttctgctgcactcaggggcccc tctccccctcggcctgtgagatctgcttcagtaggct tttctccctgactcctccatccctgtccttacaggca gctgcttctctccgggacacgaggggtccatacggac actctctactggctgggttgcgcctaactcgtgattc ctcctctgtttcag ATTCGGAGCCGGGTTGATGTCATCAGACACGTGGTAA AGAATGGTCTGCTCTGGGATGACTTGTACATAGGATT CCAAACCCGGCTTCAGCGGGATCCTGATATATACCAT CATCT gtaagtccgaaaatgcctgtcgtgtgtgccttaggct gctgcggaggaggccagggctatataagcagagtcag tgactgactgtgccctgcagtgttgatggccatggag attccaccgttagagcttttttctttgttaaccttga aggcaaatctggttaggaagataactttcaaagagtc accatctggacattcatgcccatgtgcttcaatcctg tatacaagcagtttagagtacagggaagggaaggaca ttatgaaagggagagggtgtgtttggatccagcagct ccatcctcagaatttatctgaagacactgcaaaatta ctaagaatcactatgacaagaatgaggatggggtgat atggcaaagttgtgatcctggaagaccttcatctccc atgttgcccaactctgaacatgaatttggtgaactag ttggttaaggggatgatcctccaagtttctccctggt tgagctccaaaaaccatgtaagtttctcatagcaaaa ccgtataggtccttagggctttagttggaatatttgt gctgaaatgctggaaagccccatttgccatttttgta tttgcaaaataatcatcaagaggggagaatgcattct ttcatgaccactgaccctctgaaaaggtcaggaattt agtctgaagtaggcaagcctcctaccccgcttctgcc atgagcttgcacgcacaggcctgtcttgacatttctt ctttatagatttctttttgaatatcttgaaattgctt taaaaatatttaaagaatgtagaattatataaaataa aaaggaaataaccccacacctcccacaaaaccctgtt tcctgcctttctccaCccactctccagggtaacactt ggtaacagcatagttgtatcaccccaggcctattttt gagcatatcagcatttcaagaaatgtattttttctca ataaaacatcccttatagttgaggaggggaggttatc attcctgggttttgttttttttttttttttaatgtaa tcctggtacatcggtaatttgcattttttattcatta atatctttggtatttctagtgttgggacacacaggtc aacctcagtttttgggtttttttttttgtctttttgt ctttctagggccacacctgcagcatatggacgttccc aagctaggagtctaatcagagctgtagccaccagcct acgtcatagccatagcaacgtcagatccaagccgtgt ctgtgacctacaagcacagctcatggcaacaccggat ccttaaccactgaacgaggccaggggatcgaacacac atcctcatggatcctagtcatgttcattaaccactga gtcatgatgggaactccaacttcaactattttaatgt ctgtaaaacattccatttggaaaccatttcatttgta aagcaaaatgaaaacattttgttcattttcaacagag ttcgtagctgacttctgttctggaaaaaaggaaatgg agcaaatttgagtgagaaagattcaaagataactttt cttttaaaaaaaattatatcttggaaacttctgggct attgattctgaagactatttttctatatactgttttg atagcaaagttcataaatgtgaaaggatcctgcgatg aatcttgggaagcagtcatagcccaatatatctttgt tgcttttaaaatgagatttagtttactaaatattttt ctgatcataaaaataacacagatctaccgcagaaaat ttggaaaaaaaaaaacttttaaattcaaaaaacagtt aaaccacaaatgatcccaccatccagagagcaatttg tactttggtgtctagttcatctttctttttctgttta caagcacatataccacaagcattttttcaaaaaatga aaatgggataatactatacatacgtctgtacacctgc atagttactgaacagtctttgatctaccctgtaagtt tctaacttttcattatttgaaatgatgttttggcaaa gaaatatgtaggtgtgtctcgcacactttcataatga tttcttaggataaatttcttaggataaattcataatg atttcttataataatccatactctgccaactgatctt cagggaagccaactcgccttctcagaaataacatata acccatttacttgccctctcaccaatactaggtccta atgtttttgtgtacagattctatatttttacatacaa gaattccttaaagcaaggcatgtcacagaaaaataga aggaagacacaattgtcatgtttaaggactgcattct gtaccaaaaatgctaagttaaatgaacatctgaaaca gtacagaaacgctatctttcagggaaagctgagtacc aggtactgaacagattttggcaaatacagcaggcatg gatgtttccaaaacatgtttttctactttatctctta cag GTTTTGGAATCATTTTCAAATAAAACTCCCCCTCACA CCACCTGACTGGAAGTCCTTCCTGATGTGCTCTGGGT AGAGAGGACCTGAGCTGTCCCAG gtaaagcatcctgcaggtctgggagacactcttattc tccagcccatcacactgtgtttggcatcagaattaag caggcactatgcctatcagaaaacctgacttttgggg gaatgaaagaagctaacattacaagaatgtctgtgtt taaaaataagtcaataagggagttcccatcgtggctc agtggtaacgaaccctactagtatccattgaggacac aggttcaatatctggcctcactcagtcggctaaggat ccagtgatgccgtgagctgcagtgtaggccacagacg tggctcagatctggtgctgctgtggctatggtgtagg ccggccccctgtaactccaattcgacccctaggctgg gaacctaaaaagaccccaaaaaagtcgctttaatgaa tagtgaatacatccagcccaaagtccacagactcttt ggtctggttgtggcaaacatacagccagttaacaaac aagacaaaaattatcctaggtggtcagtgggggttca gagctgaatcctgaacactggaaggaaaacagcaacc aaatccaaatactgtatggttttgcttatatgtagaa tctaaattcaaagcaaatgagcaaaccaattgaaaca gttatggaagacaagcaggtggttgtcaggggggaga taaggggaggcaggaaagacctgggcgagggagatta agaggtaccaactttcagttgcaaaacaaatgagtca ccagtatgaaatgtgcaatgtgggaaatacaggccat aactttataatctcttttttttttttgtcttttttgc cttttctaaggctgctcccgtggcatatggaggttcc caggctaggagtccaaacagagctgtagctgccagcc tacaccagagccacagcaacacgggaaccttaacccg ctgagcaaggccagggatcgaacccgagtcctcacag atgccagtagggttcattaaccactgagccacgacag gaattccagggtctgttgtgttcttaaaacacttcca ggagagtgagtggtatgtcataagtaaacaataaatg ttaaccacaacaagcttatgaaataaacaggaaagcc atatgacctacaatcagtcattgggagaatccacaaa aggttgagcagaggatcaattccagctcacactccag ttttagattctcccctgccttaaagcatcacagacta cataatctgagctgaagaataaaaattaaaactcacc ccagtgcaaaacagaaatgaaaaagtattaaaacgag gttcatactgttgttcattagcaatatcttttattca cag GGGTGCCCAACAACATGAAAAAATCAAGAATTTATTG CTGCTACGTCAAAGCTTATACCAGAGATTATGCCTTA TAGACATTAGCAATGGATAATTATATGTTGCACTTGT GAAATGTGCACATATCCTGTTTATGAATCACCACATA GCCAGATTATCAATATTTTACTTATTTCGTAAAAAAT CCACAATTTTCCATAACAGAATCAACGTGTGCAATAG GAACAAGATTGCTATGGAAAACGAGGGTAACAGGAGG AGATATTAATCCAAGCATAGAAGAAATAGACAAATGA GGGGCCATAAGGGGAATATAGGGAAGAGAAAAAAATT AAGATGGAATTTTAAAAGGAGAATGTAAAAAATAGAT ATTTGTTCCTTAATAGGTTGATTCCTCAAATAGAGCC CATGAATATAATCAAATAGGAAGGGTTCATGACTGTT TTCAATTTTTCAAAAAGCTTTGTTGAAATCATAGACT TGCAAAACAAGGCTGTAGAGGCCACCCTAAAATGGAA AATTTCACTGGGACTGAAATTATTTTGATTCAATGAC AAAATTTGTTATTACTGCGGATTATAAACTCTAACAA ATAGCGATCTCTTTGCTTCATAAAAACATAAACACTA GCTAGTAATAAAATGAGTTCTGCAG

SEQ ID NO. 50 represents contiguous genomic sequences containing Exon 12, Intron 12, Exon 13, Intron 13, Exon 14, Intron 14, Exon 15, Intron 15, Exon 16, Intron 16, Exon 17, Intron 17, and Exon 18. Nucleotide sequences that contain at least 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000 or 20,000 contiguous nucleotides of SEQ ID NO. 50 are provided, as well as nucleotide sequences at least 80, 85, 90, 95, 98, or 99% homologous to SEQ ID NO. 50.

VIII. Oligonucleotide Probes and Primers

The present invention further provides oligonucleotide probes and primers which hybridize to the hereinabove-described sequences (SEQ ID Nos. 1, 3, 5, 7, 9-45, 46, 47, 48, 49 and 50). Oligonucleotides are provided that can be homologous to SEQ ID Nos. 1, 3, 5, 7, 9-45, 46, 47, 48, 49 and 50, and fragments thereof. Oligonucleotides that hybridize under stringent conditions to SEQ ID Nos. 1, 3, 5, 7, 9-45, 46, 47, 48, 49 and 50 and fragments thereof, are also provided. Stringent conditions describe conditions under which hybridization will occur only if there is at least about 85%, about 90%, about 95%, or at least about 98% homology between the sequences. Alternatively, the oligonucleotide can have at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 75 or 100 bases which hybridize to SEQ ID Nos. 1, 3, 5, 7, 9-45, 46, 47, 48, 49 and 50, and fragments thereof. Such oligonucleotides can be used as primers and probes to detect the sequences provided herein. The probe or primer can be at least 14 nucleotides in length, and in a preferred embodiment, are at least 15, 20, 25, 28, 30, or 35 nucleotides in length.

Given the above sequences, one of ordinary skill in the art using standard algorithms can construct oligonucleotide probes and primes that are complementary to sequences contained in Seq ID Nos. 1, 3, 5, 7, 9-45, 46, 47, 48, 49 and 50, and fragments thereof. The rules for complementary pairing are well known: cytosine (“C”) always pairs with guanine (“G”) and thymine (“T”) or uracil (“U”) always pairs with adenine (“A”). It is recognized that it is not necessary for the primer or probe to be 100% complementary to the target nucleic acid sequence, as long as the primer or probe sufficiently hybridizes and can recognize the corresponding complementary sequence. A certain degree of pair mismatch can generally be tolerated.

Oligonucleotide sequences used as the hybridizing region of a primer can also be used as the hybridizing region of a probe. Suitability of a primer sequence for use as a probe depends on the hybridization characteristics of the primer. Similarly, an oligonucleotide used as a probe can be used as a primer.

It will be apparent to those skilled in the art that, provided with these specific embodiments, specific primers and probes can be prepared by, for example, the addition of nucleotides to either the 5′ or 3′ ends, which nucleotides are complementary to the target sequence or are not complimentary to the target sequence. So long as primer compositions serve as a point of initiation for extension on the target sequences, and so long as the primers and probes comprise at least 14 consecutive nucleotides contained within the above mentioned SEQ ID Nos. such compositions are within the scope of the invention.

The probes and primers herein can be selected by the following criteria, which are factors to be considered, but are not exclusive or determinative. The probes and primers are selected from the region of the CMP-Neu5Ac hydroxylase nucleic acid sequence identified in SEQ ID Nos. 1, 3, 5, 7, 945, 46, 47, 48, 49, 50, and fragments thereof. The probes and primers lack homology with sequences of other genes that would be expected to compromise the test. The probes or primers lack secondary structure formation in the amplified nucleic acid which can interfere with extension by the amplification enzyme such as E. coli DNA polymerase, preferably that portion of the DNA polymerase referred to as the Klenow fragment. This can be accomplished by employing up to about 15% by weight, preferably 5-10% by weight, dimethyl sulfoxide (DMSO) in the amplification medium and/or increasing the amplification temperatures to 30°-40° C.

Preferably, the probes or primers should contain approximately 50% guanine and cytosine nucleotides, as measured by the formula adenine (A)+thymine (T)+cytosine (C)+guanine (G)/cytosine (C)+guanine (G). Preferably, the probe or primer does not contain multiple consecutive adenine and thymine residues at the 3′ end of the primer which can result in less stable hybrids.

The probes and primers of the invention can be about 10 to 30 nucleotides long, preferably at least 10, 11, 12, 13, 14, 15, 20, 25, or 28 nucleotides in length, including specifically 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. The nucleotides as used in the present invention can be ribonucleotides, deoxyribonucleotides and modified nucleotides such as inosine or nucleotides containing modified groups which do not essentially alter their hybridization characteristics. Probe and primer sequences are represented throughout the specification as single stranded DNA oligonucleotides from the 5′ to the 3′ end. Any of the probes can be used as such, or in their complementary form, or in their RNA form (wherein T is replaced by U).

The probes and primers according to the invention can be prepared by cloning of recombinant plasmids containing inserts including the corresponding nucleotide sequences, optionally by cleaving the latter out from the cloned plasmids upon using the adequate nucleases and recovering them, e.g. by fractionation according to molecular weight. The probes and primers according to the present invention can also be synthesized chemically, for instance by the conventional phosphotriester or phosphodiester methods or automated embodiments thereof. In one such automated embodiment diethylphosphoramidites are used as starting materials and can be synthesized as described by Beaucage, et al., Tetrahedron Letters 22:1859-1862 (1981). One method of synthesizing oligonucleotides on a modified solid support is described in U.S. Pat. No. 4,458,066. It is also possible to use a probe or primer which has been isolated from a biological source (such as a restriction endonuclease digest).

The oligonucleotides used as primers or probes can also comprise nucleotide analogues such as phosphorothioates (Matsukura S., Naibunpi Gakkai Zasshi. 43(6):527-32 (1967)), alkylphosphorothiates (Miller P., et al., Biochemistry 18(23):5134-43 (1979), peptide nucleic acids (Nielsen P., et al., Science 254(5037):1497-500 (1991); Nielsen P., et al., Nucleic-Acids-Res. 21(2):197-200 (1993)), morpholino nucleic acids, locked nucleic acids, pseudocyclic oligonucleobases, 2′-O,4′-C-ethylene bridged nucleic acids or can contain intercalating agents (Asseline J., et al., Proc. Natl. Acad. Sci. USA 81(11):3297-301 (1984)).

For designing probes and primers with desired characteristics, the following useful guidelines known to the person skilled in the art can be applied. Because the extent and specificity of hybridization reactions are affected by a number of factors, manipulation of one or more of those factors will determine the exact sensitivity and specificity of a particular probe, whether perfectly complementary to its target or not. The importance and effect of various assay conditions, explained further herein, are known to those skilled in the art.

The stability of the probe and primer to target nucleic acid hybrid should be chosen to be compatible with the assay conditions. This can be accomplished by avoiding long AT-rich sequences, by terminating the hybrids with GC base pairs, and/or by designing the probe with an appropriate Tm. The beginning and end points of the probe should be chosen so that the length and % GC result in a Tm about 2-10° C. higher than the temperature at which the final assay will be performed. The base composition of the probe is significant because G-C base pairs exhibit greater thermal stability compared to A-T base pairs due to additional hydrogen bonding. Thus, hybridization involving complementary nucleic acids of higher G-C content will be stable at higher temperatures. Conditions such as ionic strength and incubation temperature under which probe will be used should also be taken into account when designing a probe. It is known that hybridization will increase as the ionic strength of the reaction mixture increases, and that the thermal stability of the hybrids will increase with increasing ionic strength. Chemical reagents, such as formamide, urea, DIVISO and alcohols, which disrupt hydrogen bonds, will increase the stringency of hybridization. Destabilization of the hydrogen bonds by such reagents can greatly reduce the Tm. In general, optimal hybridization for synthetic oligonucleotide probes of about 10-50 bases in length occurs approximately 5° C. below the melting temperature for a given duplex. Incubation at temperatures below the optimum can allow mismatched base sequences to hybridize and can therefore result in reduced specificity. It is desirable to have probes which hybridize only under conditions of high stringency. Under high stringency conditions only highly complementary nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. The degree of stringency is chosen such as to maximize the difference in stability between the hybrid formed with the target and the non-target nucleic acid. In the present case, single base pair changes need to be detected, which requires conditions of very high stringency.

The length of the target nucleic acid sequence and, accordingly, the length of the probe sequence can also be important. In some cases, there can be several sequences from a particular region, varying in location and length, which will yield probes and primers with the desired hybridization characteristics. In other cases, one sequence can be significantly better than another which differs merely by a single base.

While it is possible for nucleic acids that are not perfectly complementary to hybridize, the longest stretch of perfectly complementary base sequence will normally primarily determine hybrid stability. While oligonucleotide probes and primers of different lengths and base composition can be used, preferred oligonucleotide probes and primers of this invention are between about 14 and 30 bases in length and have a sufficient stretch in the sequence which is perfectly complementary to the target nucleic acid sequence.

Regions in the target DNA or RNA which are known to form strong internal structures inhibitory to hybridization are less preferred. Likewise, probes with extensive self-complementarity should be avoided. As explained above, hybridization is the association of two single strands of complementary nucleic acids to form a hydrogen bonded double strand. It is implicit that if one of the two strands is wholly or partially involved in a hybrid, it will be less able to participate in formation of a new hybrid. There can be intramolecular and intermolecular hybrids formed within the molecules of one type of probe if there is sufficient self complementarity. Such structures can be avoided through careful probe design. By designing a probe so that a substantial portion of the sequence of interest is single stranded, the rate and extent of hybridization can be greatly increased. Computer programs are available to search for this type of interaction. However, in certain instances, it may not be possible to avoid this type of interaction.

Specific primers and sequence specific oligonucleotide probes can be used in a polymerase chain reaction that enables amplification and detection of CMP-Neu5Ac hydroxylase nucleic acid sequences.

IV. Genetic Targeting of the CMP-Neu5Ac Hydroxylase Gene

Gene targeting allows for the selective manipulation of animal cell genomes. Using this technique, a particular DNA sequence can be targeted and modified in a site-specific and precise manner. Different types of DNA sequences can be targeted for modification, including regulatory regions, coding regions and regions of DNA between genes. Examples of regulatory regions include: promoter regions, enhancer regions, terminator regions and introns. By modifying these regulatory regions, the timing and level of expression of a gene can be altered. Coding regions can be modified to alter, enhance or eliminate the protein within a cell. Introns and exons, as well as inter-genic regions, are suitable targets for modification.

Modifications of DNA sequences can be of several types, including insertions, deletions, substitutions, or any combination thereof. A specific example of a modification is the inactivation of a gene by site-specific integration of a nucleotide sequence that disrupts expression of the gene product, i.e. a “knock out”. For example, one approach to disrupting the CMP-Neu5Ac hydroxylase gene is to insert a selectable marker into the targeting DNA such that homologous recombination between the targeting DNA and the target DNA can result in insertion of the selectable marker into the coding region of the target gene. For example, see FIGS. 3, 12, and 13. In this way, for example, the CMP-Neu5Ac hydroxylase gene sequence is disrupted, rendering the encoded enzyme nonfunctional.

Homologous Recombination

Homologous recombination permits site-specific modifications in endogenous genes and thus novel alterations can be engineered into the genome. A primary step in homologous recombination is DNA strand exchange, which involves a pairing of a DNA duplex with at least one DNA strand containing a complementary sequence to form an intermediate recombination structure containing heteroduplex DNA (see, for example Radding, C. M. (1982) Ann. Rev. Genet. 16: 405; U.S. Pat. No. 4,888,274). The heteroduplex DNA can take several forms, including a three DNA strand containing triplex form wherein a single complementary strand invades the DNA duplex (Hsieh, et al., Genes and Development 4: 1951 (1990); Rao, et al., (1991) PNAS 88:2984)) and, when two complementary DNA strands pair with a DNA duplex, a classical Holliday recombination joint or chi structure (Holliday, R., Genet. Res. 5: 282 (1964)) can form, or a double-D loop (“Diagnostic Applications of Double-D Loop Formation” U.S. Ser. No. 07/755,462, filed Sep. 4, 1991). Once formed, a heteroduplex structure can be resolved by strand breakage and exchange, so that all or a portion of an invading DNA strand is spliced into a recipient DNA duplex, adding or replacing a segment of the recipient DNA duplex. Alternatively, a heteroduplex structure can result in gene conversion, wherein a sequence of an invading strand is transferred to a recipient DNA duplex by repair of mismatched bases using the invading strand as a template (Genes, 3^(rd) Ed. (1987) Lewin, B., John Wiley, New York, N.Y.; Lopez, et al., Nucleic Acids Res. 15: 5643 (1987)). Whether by the mechanism of breakage and rejoining or by the mechanism(s) of gene conversion, formation of heteroduplex DNA at homologously paired joints can serve to transfer genetic sequence information from one DNA molecule to another.

The ability of homologous recombination (gene conversion and classical strand breakage/rejoining) to transfer, genetic sequence information between DNA molecules renders targeted homologous recombination a powerful method in genetic engineering and gene manipulation.

In homologous recombination, the incoming DNA interacts with and integrates into a site in the genome that contains a substantially homologous DNA sequence. In non-homologous (“random” or “illicit”) integration, the incoming DNA is not found at a homologous sequence in the genome but integrates elsewhere, at one of a large number of potential locations. In general, studies with higher eukaryotic cells have revealed that the frequency of homologous recombination is far less than the frequency of random integration. The ratio of these frequencies has direct implications for “gene targeting” which depends on integration via homologous recombination (i.e. recombination between the exogenous “targeting DNA” and the corresponding “target DNA” in the genome).

A number of papers describe the use of homologous recombination in mammalian cells. Illustrative of these papers are Kucherlapati, et al., Proc. Natl. Acad. Sci. (USA) 81:3153-3157, 1984; Kucherlapati, et al., Mol. Cell. Bio. 5:714-720, 1985; Smithies, et al, Nature 317:230-234, 1985; Wake, et al., Mol. Cell. Bio. 8:2080-2089, 1985; Ayares, et al., Genetics 111:375-388, 1985; Ayares, et al., Mol. Cell. Bio. 7:1656-1662, 1986; Song, et al., Proc. Natl. Acad. Sci. USA 84:6820-6824, 1987; Thomas, et al. Cell 44:419-428, 1986; Thomas and Capecchi, Cell 51: 503-512, 1987; Nandi, et al., Proc. Natl. Acad. Sci. USA 85:3845-3849, 1988; and Mansour, et al., Nature 336:348-352, 1988; Evans and Kaufman, Nature 294:146-154, 1981; Doetschman, et al., Nature 330:576-578, 1987; Thoma and Capecchi, Cell 51:503-512, 4987; Thompson, et al., Cell 56:316-321, 1989.

The present invention uses homologous recombination to inactivate the porcine CMP-Neu5Ac hydroxylase gene in cells, such as fibroblasts. The DNA can comprise at least a portion of the gene(s) at the particular locus with introduction of an alteration into at least one, optionally both copies, of the native gene(s), so as to prevent expression of a functional enzyme and production of a Hanganutziu-Deicher antigen molecule. The alteration can be an insertion, deletion, replacement or combination thereof. When the alteration is introduce into only one copy of the gene being inactivated, the cells having a single unmutated copy of the target gene are amplified and can be subjected to a second targeting step, where the alteration can be the same or different from the first alteration, usually different, and where a deletion, or replacement is involved, can be overlapping at least a portion of the alteration originally introduced. In this second targeting step, a targeting vector with the same arms of homology, but containing a different mammalian selectable markers can be used. The resulting transformants are screened for the absence of a functional target antigen and the DNA of the cell can be further screened to ensure the absence of a wild-type target gene. Alternatively, homozygosity as to a phenotype can be achieved by breeding hosts heterozygous for the mutation.

Porcine cells that can be genetically modified can be obtained from a variety of different organs and tissues such as, but not limited to, brain, heart, lungs, glands, brain, eye, stomach, spleen, pancreas, kidneys, liver, intestines, uterus, bladder, skin, hair, nails, ears, nose, mouth, lips, gums, teeth, tongue, salivary glands, tonsils, pharynx, esophagus, large intestine, small intestine, rectum, anus, pylorus, thyroid gland, thymus gland, suprarenal capsule, bones, cartilage, tendons, ligaments, skeletal muscles, smooth muscles, blood vessels, blood, spinal cord, trachea, ureters, urethra, hypothalamus, pituitary, adrenal glands, ovaries, oviducts, uterus, vagina, mammary glands, testes, seminal vesicles, penis, lymph, lymph nodes and lymph vessels. In one embodiment of the invention, porcine cells can be selected from the group consisting of, but not limited to, epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, □hosphate cells, cumulus cells, epidermal cells, endothelial cells, Islets of Langerhans cells, blood cells, blood precursor cells, bone cells, bone precursor cells, neuronal stem cells, primordial stem cells, hepatocytes, keratinocytes, umbilical vein endothelial cells, aortic endothelial cells, microvascular endothelial cells, fibroblasts, liver stellate cells, aortic smooth muscle cells, cardiac myocytes, neurons, Kupffer cells, smooth muscle cells, Schwann cells, and epithelial cells, erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes, pancreatic islet cells, thyroid cells, parathyroid cells, parotid cells, tumor cells, glial cells, astrocytes, red blood cells, white blood cells, macrophages, epithelial cells, somatic cells, pituitary cells, adrenal cells, hair cells, bladder cells, kidney cells, retinal cells, rod cells, cone cells, heart cells, pacemaker cells, spleen cells, antigen presenting cells, memory cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, uterine cells, prostate cells, vaginal epithelial cells, sperm cells, testicular cells, germ cells, egg cells, leydig cells, peritubular cells, sertoli cells, lutein cells, cervical cells, endometrial cells, mammary cells, follicle cells, mucous cells, ciliated cells, nonkeratinized epithelial cells, keratinized epithelial cells, lung cells, goblet cells, columnar epithelial cells, squamous epithelial cells, osteocytes, osteoblasts, and osteoclasts.

In one alternative embodiment, embryonic stem cells can be used. An embryonic stem cell line can be employed or embryonic stem cells can be obtained freshly from a host, such as a porcine animal. The cells can be grown on an appropriate fibroblast-feeder layer or grown in the presence of leukemia inhibiting factor (LIF). In a preferred embodiment, the porcine cells can be fibroblasts; in one specific embodiment, the porcine cells can be fetal fibroblasts. Fibroblast cells are a preferred somatic cell type because they can be obtained from developing fetuses and adult animals in large quantities.

These cells can be easily propagated in vitro with a rapid doubling time and can be clonally propagated for use in gene targeting procedures.

Targeting Vectors

Cells homozygous at a targeted locus can be produced by introducing DNA into the cells, where the DNA has homology to the target locus and includes a marker gene, allowing for selection of cells comprising the integrated construct. The homologous DNA in the target vector will recombine with the chromosomal DNA at the target locus (see, for example, FIGS. 3, 12, and 13). The marker gene can be flanked on both sides by homologous DNA sequences, a 3′ recombination arm and a 5′ recombination arm (See, for example, FIG. 11). Methods for the construction of targeting vectors have been described in the art, see, for example, Dai et al., Nature Biotechnology 20: 251-255, 2002; WO 00/51424.

Various constructs can be prepared for homologous recombination at a target locus. Usually, the construct can include at least 50 bp, 100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp of sequence homologous with the target locus. The sequence can include any contiguous sequence of the porcine CMP-Neu5Ac hydroxylase gene, including at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90.95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 700, 750, 800, 850, 900, 1000, 5000 or 10, 000 contiguous nucleotides of Seq ID Nos 9-45, 46, 47, 48, 49, and 50, or any combination or fragment thereof. Fragments of Seq ID Nos. 9-45, 46, 47, 48, 49 and 50 can include any contiguous nucleic acid or peptide sequence that includes at least about 10 bp, 15 bp, 17 bp, 20 bp, 50 bp, 100 bp, 500 bp, 1 kbp, 5 kbp or 10 kbp.

Various considerations can be involved in determining the extent of homology of target DNA sequences, such as, for example, the size of the target locus, availability of sequences, relative efficiency of double cross-over events at the target locus and the similarity of the target sequence with other sequences.

The targeting DNA can include a sequence in which DNA substantially isogenic flanks the desired sequence modifications with a corresponding target sequence in the genome to be modified. The substantially isogenic sequence can be at least about 95%, 97-98%, 99.0-99.5%, 99.6-99.9%, or 100% identical to the corresponding target sequence (except for the desired sequence modifications). The targeting DNA and the target DNA preferably can share stretches of DNA at least about 75, 150 or 500 base pairs that are 100% identical. Accordingly, targeting DNA can be derived from cells closely related to the cell line being targeted; or the targeting DNA can be derived from cells of the same cell line or animal as the cells being targeted.

The DNA constructs can be designed to modify the endogenous, target CMP-Neu5Ac hydroxylase. The homologous sequence for targeting the construct can have one or more deletions, insertions, substitutions or combinations thereof designed to disrupt the function of the resultant gene product. In one embodiment, the alteration can be the insertion of a selectable marker gene fused in reading frame with the upstream sequence of the target gene.

Suitable selectable marker genes include, but are not limited to: genes conferring the ability to grow on certain media substrates, such as the tk gene (thymidine kinase) or the hprt gene (hypoxanthine phosphoribosyltransferase) which confer the ability to grow on HAT medium (hypoxanthine, aminopterin and thymidine); the bacterial gpt gene (guanine/xanthine phosphoribosyltransferase) which allows growth on MAX medium (mycophenolic acid, adenine, and xanthine). See, for example, Song, K-Y., et al. Proc. Nat'l Acad. Sci. U.S.A. 84:6820-6824 (1987); Sambrook, J., et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), Chapter 16. Other examples of selectable markers include: genes conferring resistance to compounds such as antibiotics, genes conferring the ability to grow on selected substrates, genes encoding proteins that produce detectable signals such as luminescence, such as green fluorescent protein, enhanced green fluorescent protein (eGFP). A wide variety of such markers are known and available, including, for example, antibiotic resistance genes such as the neomycin resistance gene (neo) (Southern, P., and P. Berg, J. Mol. Appl. Genet. 1:327-341 (1982)); and the hygromycin resistance gene (hyg) (Nucleic Acids Research 11:6895-6911 (1983), and Te Riele, H., et al., Nature 348:649-651 (1990)). Other selectable marker genes include: acetohydroxy acid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline.

Methods for the incorporation of antibiotic resistance genes and negative selection factors will be familiar to those of ordinary skill in the art (see, e.g., WO 99/15650; U.S. Pat. No. 6,080,576; U.S. Pat. No. 6,136,566; Niwa, et al., J. Biochem. 113:343-349 (1993); and Yoshida, et al., Transgenic Research, 4:277-287 (1995)).

Additional selectable marker genes useful in this invention, for example, are described in U.S. Pat. Nos. 6,319,669; 6,316,181; 6,303,373; 6,291,177; 6,284,519; 6,284,496; 6,280,934; 6,274,354; 6,270,958; 6,268,201; 6,265,548; 6,261,760; 6,255,558; 6,255,071; 6,251,677; 6,251,602; 6,251,582; 6,251,384; 6,248,558; 6,248,550; 6,248,543; 6,232,107; 6,228,639; 6,225,082; 6,221,612; 6,218,185; 6,214,567; 6,214,563; 6,210,922; 6,210,910; 6,203,986; 6,197,928; 6,180,343; 6,172,188; 6,153,409; 6,150,176; 6,146,826; 6,140,132; 6,136,539; 6,136,538; 6,133,429; 6,130,313; 6,124,128; 6,110,711; 6,096,865; 6,096,717; 6,093,808; 6,090,919; 6,083,690; 6,077,707; 6,066,476; 6,060,247; 6,054,321; 6,037,133; 6,027,881; 6,025,192; 6,020,192; 6,013,447; 6,001,557; 5,994,077; 5,994,071; 5,993,778; 5,989,808; 5,985,577; 5,968,773; 5,968,738; 5,958,713; 5,952,236; 5,948,889; 5,948,681; 5,942,387; 5,932,435; 5,922,576; 5,919,445; and 5,914,233.

Combinations of selectable markers can also be used. For example, to target CMP-Neu5Ac hydroxylase, a neo gene (with or without its own promoter, as discussed above) can be cloned into a DNA sequence which is homologous to the CMP-Neu5Ac hydroxylase gene. To use a combination of markers, the HSV-tk gene can be cloned such that it is outside of the targeting DNA (another selectable marker could be placed on the opposite flank, if desired). After introducing the DNA construct into the cells to be targeted, the cells can be selected on the appropriate antibiotics. In this particular example, those cells which are resistant to G418 and gancyclovir are most likely to have arisen by homologous recombination in which the neo gene has been recombined into the CMP-Neu5Ac hydroxylase gene but the tk gene has been lost because it was located outside the region of the double crossover.

Deletions can be at least about 50 bp, more usually at least about 100 bp, and generally not more than about 20 kbp, where the deletion can normally include at least a portion of the coding region including a portion of or one or more exons, a portion of or one or more introns, and can or can not include a portion of the flanking non-coding regions, particularly the 5′-non-coding region (transcriptional regulatory region). Thus, the homologous region can extend beyond the coding region into the 5′-non-coding region or alternatively into the 3′-non-coding region. Insertions can generally not exceed 10 kbp, usually not exceed 5 kbp, generally being at least 50 bp, more usually at least 200 bp.

The region(s) of homology can include mutations, where mutations can further inactivate the target gene, in providing for a frame shift, or changing a key amino acid, or the mutation can correct a dysfunctional allele, etc. Usually, the mutation can be a subtle change, not exceeding about 5% of the homologous flanking sequences.

The construct can be prepared in accordance with methods known in the art, various fragments can be brought together, introduced into appropriate vectors, cloned, analyzed and then manipulated further until the desired construct has been achieved (see, for example FIGS. 5-11). Various modifications can be made to the sequence, to allow for restriction analysis, excision, identification of probes, etc. Silent mutations can be introduced, as desired. At various stages, restriction analysis, sequencing, amplification with the polymerase chain reaction, primer repair, in vitro mutagenesis, etc. can be employed.

The construct can be prepared using a bacterial vector, including a prokaryotic replication system, e.g. an origin recognizable by E. coli, at each stage the construct can be cloned and analyzed. A marker, the same as or different from the marker to be used for insertion, can be employed, which can be removed prior to introduction into the target cell. Once the vector containing the construct has been completed, it can be further manipulated, such as by deletion of the bacterial sequences, linearization, introducing a short deletion in the homologous sequence. After final manipulation, the construct can be introduced into the cell.

Techniques which can be used to allow the DNA construct entry into the host cell include calcium phosphate/DNA co-precipitation, microinjection of DNA into the nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, or any other technique known by one skilled in the art. The DNA can be single or double stranded, linear or circular, relaxed or supercoiled DNA. For various techniques for transfecting mammalian cells, see, for example, Keown et al., Methods in Enzymology Vol. 185, pp. 527-537 (1990).

The present invention further includes recombinant constructs comprising one or more of the sequences as broadly described above (for example in Tables 9-12). The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. The construct can also include regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example: pBs, pQE-9 (Qiagen), phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSv2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPv, pMSG, pSVL (Pharmacia). Also, any other plasmids and vectors can be used as long as they are replicable and viable in the host. Vectors known in the art and those commercially available (and variants or derivatives thereof) can in accordance with the invention be engineered to include one or more recombination sites for use in the methods of the invention. Such vectors can be obtained from, for example, Vector Laboratories Inc., Invitrogen, Promega, Novagen, NEB, Clontech, Boehringer Mannheim, Pharmacia, EpiCenter, OriGenes Technologies Inc., Stratagene, PerkinElmer, Pharmingen, and Research Genetics. Other vectors of interest include eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.), p3′SS, pXT1, pSG5, pPbac, pMbac, pMC1neo, and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBacHis A, B, and C, pVL1392, pBlueBacIII, pCDM8, pcDNA1, pZeoSV, pcDNA3 pREP4, pCEP4, and pEBVHis (Invitrogen, Corp.) and variants or derivatives thereof.

Other vectors suitable for use in the invention include pUC18, pUC19, pBlueScript, pSPORT, cosmids, phagemids, YAC's (yeast artificial chromosomes), BAC's (bacterial artificial chromosomes), PI (Escherichia coli phage), pQE70, pQE60, pQE9 (quagan), pBS vectors, PhageScript vectors, BlueScript vectors, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene), pcDNA3 (Invitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pSPORT1, pSPORT2, pCMVSPORT2.0 and pSV-SPORT1 (Invitrogen) and variants or derivatives thereof. Viral vectors can also be used, such as lentiviral vectors (see, for example, WO 03/059923; Tiscomia et al. PNAS 100:1844-1848 (2003)).

Additional vectors of interest include pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2, pRSET, pBlueBacHis2, pcDNA3.1/His, pcDNA3.1(−)/Myc-His, pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pAO815, pPICZ, pPICZA, pPICZB, pPICZC, pGAPZA, pGAPZB, pGAPZC, pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND, pIND(SP1), pVgRXR, pcDNA2.1, pYES2, pZErO1.1, pZErO-2.1, pCR-Blunt, pSE280, pSE380, pSE420, pVL1392, pVL1393, pCDM8, pcDNA1.1, pcDNA 1.1/Amp, pcDNA3.1, pcDNA3.1/Zeo, pSe, SV2, pRc/CMV2, pRc/RSV, pREP4, pREP7, pREP8, pREP9, pREP 10, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, and pCRBac from Invitrogen; λ ExCell, λ gt11, pTrc99A, pKK223-3, pGEX-1λ T, pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX-5X-1, pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG, pCH110, pKK232-8, pSL1180, pNEO, and pUC4K from Pharmacia; pSCREEN-1b(+), pT7Blue(R), pT7Blue-2, pCITE-4abc(+), pOCUS-2, pTAg, pET-32LIC, pET-30LIC, pBAC-2 cp LIC, pBACgus-2 cp LIC, pT7Blue-2 LIC, pT7Blue-2, λ SCREEN-1, λ BlueSTAR, pET-3abcd, pET-7abc, pET9abcd, pET11abcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b-pET-17xb, pET-19b, pET-20b(+), pET-21 abcd(+), pET-22b(+), pET-23abcd(+), pET-24abcd(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28abc(+), pET-29abc(+), pET-30abc(+), pET-31b(+), pET-32abc(+), pET-33b(+), pBAC-1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3 cp, pBACgus-2 cp, pBACsurf-1, pig, Signal pig, pYX, Selecta Vecta-Neo, Selecta Vecta-Hyg, and Selecta Vecta-Gpt from Novagen; pLexA, pB42AD, pGBT9, pAS2-1, pGAD424, pACT2, pGAD GL, pGAD GH, pGAD10, pGilda, pEZM3, pEGFP, pEGFP-1, pEGFP-N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis-GFP, pSEAP2-Basic, pSEAP2-Contral, pSEAP2-Promoter, pSEAP2-Enhancer, pβgal-Basic, pβgal-Control, pβgal-Promoter, pβgal-Enhancer, pCMV, pTet-Off, pTet-On, pTK-Hyg, pRetro-Off, pRetro-On, pIRES1neo, pIRES1hyg, pLXSN, pLNCX, pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX4T-1/2/3, pYEX-S1, pBacPAK-His, pBacPAK8/9, pAcUW31, BacPAK6, pTriplEx, λgt10, λgt11, pWE15, and λTriplEx from Clontech; Lambda ZAP II, pBK-CMV, pBK-RSV, pBluescript II KS +/−, pBluescript II SK +/−, pAD-GAL4, pBD-GAL4 Cam, pSurfscript, Lambda FIX II, Lambda DASH, Lambda EMBL3, Lambda EMBL4, SuperCos, pCR-Script Amp, pCR-Script Cam, pCR-Script Direct, pBS +/−, pBC KS +/−, pBC SK +/−, Phagescript, pCAL-n-EK, pCAL-n, pCAL-c, pCAL-kc, pET-3abcd, pET-11abcd, pSPUTK, pESP-1, pCMVLacI, pOPRSVI/MCS, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pMC1neo Poly A, pOG44, pOG45, pFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414, pRS415, and pRS416 from Stratagene.

Additional vectors include, for example, pPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGAD1-3, pGAD10, pACt, pACT2, pGADGL, pGADGH, pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA, pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5, pNLexA, pYESTrp and variants or derivatives thereof.

Also, any other plasmids and vectors known in the art can be used as long as they are replicable and viable in the host.

Selection of Homologously Recombined Cells

Cells that have been homologously recombined to knock-out expression of the porcine CMP-Neu5Ac hydroxylase gene can then be grown in appropriately-selected medium to identify cells providing the appropriate integration. Those cells which show the desired phenotype can then be further analyzed by restriction analysis, electrophoresis, Southern analysis, polymerase chain reaction, or another technique known in the art. By identifying fragments which show the appropriate insertion at the target gene site, cells can be identified in which homologous recombination has occurred to inactivate or otherwise modify the target gene.

The presence of the selectable marker gene inserted into the CMP-Neu5Ac hydroxylase gene establishes the integration of the target construct into the host genome. Those cells which show the desired phenotype can then be further analyzed by restriction analysis, electrophoresis, Southern analysis, polymerase chain reaction, monoclonal antibody assays, Fluorescent Activated Cell Sorter (FACS), or any other techniques or methods known in the art to analyze the DNA in order to establish whether homologous or non-homologous recombination occurred. This can be determined by employing probes for the insert and then sequencing the 5′ and 3′ regions flanking the insert for the presence of the CMP-Neu5Ac hydroxylase gene extending beyond the flanking regions of the construct or identifying the presence of a deletion, when such deletion is introduced. Primers can also be used which are complementary to a sequence within the construct and complementary to a sequence outside the construct and at the target locus. In this way, one can only obtain DNA duplexes having both of the primers present in the complementary chains if homologous recombination has occurred. By demonstrating the presence of the primer sequences or the expected size sequence, the occurrence of homologous recombination is supported.

The polymerase chain reaction used for screening homologous recombination events is described, for example, in Kim and Smithies, Nucleic Acids Res. 16:8887-8903, 1988; and Joyner, et al., Nature 338:153-156, 1989.

An alternative method for screening homologous recombination events includes utilizing monoclonal or polyclonal antibodies specific for porcine CMP-Neu5Ac Hydroxylase and/or Neu5Gc, as described in, for example, Malykh, et al., European Journal of Cell Biology 80, 48-58 (2001), Malykh, et al., Glycoconjugate J. 15, 885-893 (1998).

Further characterization of porcine cells lacking expression of functional CMP-Neu5Ac Hydroxylase due to homologous recombination events include, but are not limited to, Southern Blot analysis, Northern Blot analysis, specific lectin binding assays, and/or sequence analysis, or by using anti-Neu5Gc or anti-CMP-Neu5Ac hydroxylase antibody assays as described, for example, in Y. Malykh, et. al. Biochem J. 370: 601-607 (2003); Y. Malykh, et al. European Journal of Cell Biology 80: 48-58 (2001); Y. Malykh et al. Glycoconjugate J. 15: 885-893 (1998). See generally, for example, A. Sharma, et al. Transplantation 75(4): 430-436 (2003).

The cell lines obtained from the first round of targeting are likely to be heterozygous for the targeted allele. Homozygosity, in which both alleles are modified, can be achieved in a number of ways. One approach is to grow up a number of cells in which one copy has been modified and then to subject these cells to another round of targeting of the remaining porcine CMP-Neu5Ac hydroxylase allele using a different selectable marker. Alternatively, homozygotes can be obtained by breeding animals heterozygous for the modified allele, according to traditional Mendelian genetics. In some situations, it can be desirable to have two different modified alleles. This can be achieved by successive rounds of gene targeting or by breeding heterozygotes, each of which carries one of the desired modified alleles.

VIII. Genetic Manipulation of Additional Genes to Overcome Immunologic Barriers of Xenotransplantation

In one aspect of the invention, cells homozygous for the nonfunctional CMP-Neu5Ac hydroxylase gene can be subject to further genetic modification. For example, one can introduce additional genetic capability into the homozygotic hosts, where the endogenous CMP Neu5Ac hydroxylase alleles have been made nonfunctional, to substitute, replace or provide different genetic capability to the host. One can remove the marker gene after homogenization. By introducing a construct comprising substantially the same homologous DNA, possibly with extended sequences, having the marker gene portion of the original construct deleted, one can be able to obtain homologous recombination with the target locus. By using a combination of marker genes for integration, one providing positive selection and the other negative selection, in the removal step, one can select against the cells retaining the marker genes.

In one embodiment, porcine cells are provided that lack the CMP-Neu5Ac hydroxylase gene and the α(1,3)GT gene. Animals lacking functional CMP-Neu5Ac hydroxylase can be produced according to the present invention, and then cells from this animal can be used to knockout the α(1,3)GT gene. Homozygous α(1,3)GT negative porcine have recently been reported (Phelps et. al. Science 2003; WO 04/028243). Alternatively, cells from these a(1,3)GT knockout animals can be used and further modified to inactivate the CMP-Neu5Ac hydroxylase gene.

In another embodiment, porcine cells are also provided that lack the porcine CMP-Neu5Ac hydroxylase gene and produce human complement inhibiting proteins. Animals lacking functional porcine CMP-Neu5Ac hydroxylase gene can be produced according to the present invention, and then cells from this animal can be further modified to express human complement inhibiting proteins, such as, but not limited to, CD59 (cDNA reported by Philbrick, W. M., et al. (1990) Eur. J. Immunol. 20:87-92), human decay accelerating factor (DAF) (cDNA reported by Medof, et al. (1987) Proc. Natl. Acad. Sci. USA 84: 2007), and human membrane cofactor protein (MCP) (cDNA reported by Lublin, D., et al. (1988) J. Exp. Med. 168: 181-194).

In an alternative embodiment, cells from transgenic pigs producing human complement inhibiting proteins can be used and further modified to inactivate the porcine CMP-Neu5Ac hydroxylase gene. Transgenic pigs producing human complement inhibiting proteins are known in the art (see, for example, U.S. Pat. No. 6,166,288).

In a further embodiment, porcine cells are provided that lack the porcine CMP-Neu5Ac hydroxylase gene and the porcine Forssman synthetase (FSM) gene. Animals lacking functional porcine CMP-Neu5Ac hydroxylase gene can be produced according to the present invention, and then cells from this animal can be further modified to knockout the porcine FSM synthetase gene, which is involved in the production of gal-α-gal epitopes, and plays a role in xenotransplant rejection. The porcine FSM synthetase gene has recently been identified (see U.S. Application 60/568,922). Alternatively, cells from these FSM synthetase gene knockout animals can be used and further modified to inactivate the porcine CMP-Neu5Ac hydroxylase gene.

In a still further embodiment, porcine cells are provided that lack the porcine CMP-Neu5Ac hydroxylase gene and the porcine isogloboside 3 synthase gene. Animals lacking functional porcine CMP-Neu5Ac hydroxylase gene can be produced according to the present invention, and then cells from this animal can be used to knockout the porcine iGb3 synthase gene. The porcine iGb3 synthase gene has recently been reported (U.S. Application No. 60/517,524). Alternatively, cells from these porcine iGb3 synthase gene knockout animals can be used and further modified to inactivate the porcine CMP-Neu5Ac hydroxylase gene.

In another embodiment, porcine cells are provided that lack the porcine CMP-Neu5Ac hydroxylase gene, the α(1,3)GT gene, the FSM synthetase gene, and the porcine iGb3 synthase gene. Animals lacking functional CMP-Neu5Ac hydroxylase gene can be produced according to the present invention, and then cells from this animal can be used to knockout the α(1,3)GT gene, the FSM synthetase gene, and the porcine iGb3 synthase gene. Homozygous α(1,3)GT-negative porcine have recently been reported (Phelps et al. supra, Science 2003; WO 04/028243) Alternatively, cells from these a(1,3)GT knockout animals can be used and further modified to inactivate the porcine iGb3 synthase gene, the porcine FSM synthetase gene, and the CMP-Neu5Ac hydroxylase gene, and, in addition, express human complement inhibiting proteins, such as, but not limited to, CD59, human decay accelerating factor (DAF), and human membrane cofactor protein (MCP).

VIII. Production of Genetically Modified Animals

The present invention provides methods of producing a transgenic pig that lacks expression of CMP-Neu5Ac hydroxylase through the genetic modification of porcine totipotent embryonic cells. In one embodiment, the animals can be produced by: (a) identifying one or more target CMP-Neu5Ac hydroxylase nucleic acid genomic sequences in an animal; (b) preparing one or more homologous recombination vectors targeting the CMP-Neu5Ac hydroxylase nucleic acid genomic sequences; (c) inserting the one or more targeting vectors into the genomes of a plurality of totipotent cells of the animal, thereby producing a plurality of transgenic totipotent cells; (d) obtaining a tetraploid blastocyst of the animal; (e) inserting the plurality of totipotent cells into the tetraploid blastocyst, thereby producing a transgenic embryo; (f) transferring the embryo to a recipient female animal; and (g) allowing the embryo to develop to term in the female animal. The method of transgenic animal production described here by which to generate a transgenic pig is further generally described in U.S. Pat. No. 6,492,575.

In another embodiment, the totipotent cells can be embryonic stem (ES) cells. The isolation of ES cells from blastocysts, the establishing of ES cell lines and their subsequent cultivation are carried out by conventional methods as described, for example, by Doetchmann et al., J. Embryol. Exp. Morph. 87:27-45 (1985); Li et al., Cell 69:915-926 (1992); Robertson, E. J. “Tetracarcinomas and Embryonic Stem Cells: A Practical Approach,” ed. E. J. Robertson, IRL Press, Oxford, England (1987); Wurst and Joyner, “Gene Targeting: A Practical Approach,” ed. A. L. Joyner, IRL Press, Oxford, England (1993); Hogen et al., “Manipulating the Mouse Embryo: A Laboratory Manual,” eds. Hogan, Beddington, Costantini and Lacy, Cold Spring Harbor Laboratory Press, New York (1994); and Wang, et al., Nature 336:741-744 (1992). For example, after transforming embryonic stem cells with the targeting vector to alter the CMP-Neu5Ac hydroxylase gene, the cells can be plated onto a feeder layer in an appropriate medium, for example, such as fetal bovine serum enhanced DMEM. Cells containing the construct can be detected by employing a selective medium, and after sufficient time for colonies to grow, colonies can be picked and analyzed for the occurrence of homologous recombination. Polymerase chain reaction can be used, with primers within and without the construct sequence but at the target locus. Those colonies which show homologous recombination can then be used for embryo manipulating and blastocyst injection. Blastocysts can be obtained from superovulated females. The embryonic stem cells can then be trypsinized and the modified cells added to a droplet containing the blastocysts. At least one of the modified embryonic stem cells can be injected into the blastocoel of the blastocyst. After injection, at least one of the blastocysts can be returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting litters screened for mutant cells having the construct. The blastocysts are selected for different parentage from the transformed ES cells. By providing for a different phenotype of the blastocyst and the ES cells, chimeric progeny can be readily detected, and then genotyping can be conducted to probe for the presence of the modified CMP-Neu5Ac hydroxylase gene.

In a further embodiment of the invention, the totipotent cells can be embryonic germ (EG) cells. Embryonic Germ cells are undifferentiated cells functionally equivalent to ES cells, that is they can be cultured and transfected in vitro, then contribute to somatic and germ cell lineages of a chimera (Stewart et al., Dev. Biol. 161:626-628 (1994)). EG cells are derived by culture of primordial germ cells, the progenitors of the gametes, with a combination of growth factors: leukemia inhibitory factor, steel factor and basic fibroblast growth factor (Matsui, et al., Cell 70:841-847 (1992); Resnick, et al., Nature 359:550-551 (1992)). The cultivation of EG cells can be carried out using methods known to one skilled in the art, such as described in Donovan et al., “Transgenic Animals, Generation and Use,” Ed. L. M. Houdebine, Harwood Academic Publishers (1997).

Tetraploid blastocysts for use in the invention can be obtained by natural zygote production and development, or by known methods by electrofusion of two-cell embryos and subsequently cultured as described, for example, by James, et al., Genet. Res. Camb. 60:185-194 (1992); Nagy and Rossant, “Gene Targeting: A Practical Approach,” ed. A. L. Joyner, IRL Press, Oxford, England (1993); or by Kubiak and Tarkowski, Exp. Cell Res. 157:561-566 (1985).

The introduction of the ES cells or EG cells into the blastocysts can be carried out by any method known in the art, for example, as described by Wang, et al., EMBO J. 10:2437-2450 (1991).

A “plurality” of totipotent cells can encompass any number of cells greater than one. For example, the number of totipotent cells for use in the present invention can be about 2 to about 30 cells, about 5 to about 20 cells, or about 5 to about 10 cells. In one embodiment, about 5-10 ES cells taken from a single cell suspension are injected into a blastocyst immobilized by a holding pipette in a micromanipulation apparatus. Then the embryos are incubated for at least 3 hours, possibly overnight, prior to introduction into a female recipient animal via methods known in the art (see for example Robertson, E. J. “Teratocarcinomas and Embryonic Stem Cells: A Practical Approach” IRL Press, Oxford, England (1987)). The embryo can then be allowed to develop to term in the female animal.

Somatic Cell Nuclear Transfer to Produce Cloned, Transgenic Offspring

The present invention provides a method for cloning a pig lacking a functional CMP-Neu5Ac hydroxylase gene via somatic cell nuclear transfer. In general, a wide variety of methods to accomplish mammalian cloning are currently being rapidly developed and reported, any method that accomplishes the desired result can be used in the present invention. Nonlimiting examples of such methods are described below. For example, the pig can be produced by a nuclear transfer process comprising the following steps: obtaining desired differentiated pig cells to be used as a source of donor nuclei; obtaining oocytes from a pig; enucleating the oocytes; transferring the desired differentiated cell or cell nucleus into the enucleated oocyte, e.g., by fusion or injection, to form NT units; activating the resultant NT unit; and transferring said cultured NT unit to a host pig such that the NT unit develops into a fetus.

Nuclear transfer techniques or nuclear transplantation techniques are known in the art (Campbell et al, Theriogenology, 43:181 (1995); Collas, et al, Mol. Report. Dev., 38:264-267 (1994); Keefer et al, Biol. Reprod., 50:935-939 (1994); Sims, et al, Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993); WO 94/26884; WO 94/24274, and WO 90/03432, U.S. Pat. Nos. 4,944,384 and 5,057,420). In one nonlimiting example, methods are provided such as those described in U.S. Patent Publication No. 2003/0046722 to Collas, et al., which describes methods for cloning mammals that allow the donor chromosomes or donor cells to be reprogrammed prior to insertion into an enucleated oocyte. The invention also describes methods of inserting or fusing chromosomes, nuclei or cells with oocytes.

A donor cell nucleus, which has been modified to alter the CMP-Neu5Ac hydroxylase gene, is transferred to a recipient porcine oocyte. The use of this method is not restricted to a particular donor cell type. The donor cell can be as described in Wilmut, et al., Nature 385 810 (1997); Campbell, et al., Nature 380 64-66 (1996); or Cibelli, et al., Science 280 1256-1258 (1998). All cells of normal karyotype, including embryonic, fetal and adult somatic cells which can be used successfully in nuclear transfer can in principle be employed. Fetal fibroblasts are a particularly useful class of donor cells. Generally suitable methods of nuclear transfer are described in Campbell, et al., Theriogenology 43 181 (1995), Collas, et al., Mol. Reprod. Dev. 38 264-267 (1994), Keefer, et al., Biol. Reprod. 50 935-939 (1994), Sims, et al., Proc. Nat'l. Acad. Sci. USA 90 6143-6147 (1993), WO-A-9426884, WO-A-9424274, WO-A-9807841, WO-A-9003432, U.S. Pat. No. 4,994,384 and U.S. Pat. No. 5,057,420. Differentiated or at least partially differentiated donor cells can also be used. Donor cells can also be, but do not have to be, in culture and can be quiescent. Nuclear donor cells which are quiescent are cells which can be induced to enter quiescence or exist in a quiescent state in vivo. Prior art methods have also used embryonic cell types in cloning procedures (Campbell, et al. (Nature, 380:64-68, 1996) and Stice, et al (Biol. Reprod., 20 54:100-110, 1996).

Somatic nuclear donor cells may be obtained from a variety of different organs and tissues such as, but not limited to, skin, mesenchyme, lung, pancreas, heart, intestine, stomach, bladder, blood vessels, kidney, urethra, reproductive organs, and a disaggregated preparation of a whole or part of an embryo, fetus or adult animal. In a suitable embodiment of the invention, nuclear donor cells are selected from the group consisting of epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, granulose cells, cumulus cells, epidermal cells or endothelial cells. In another embodiment, the nuclear cell is an embryonic stem cell. In a preferred embodiment, fibroblast cells can be used as donor cells.

In another embodiment of the invention, the nuclear donor cells of the invention are germ cells of an animal. Any germ cell of an animal species in the embryonic, fetal, or adult stage may be used as a nuclear donor cell. In a suitable embodiment, the nuclear donor cell is an embryonic germ cell.

Nuclear donor cells may be arrested in any phase of the cell cycle (GO, GI, G2, S, M) so as to ensure coordination with the acceptor cell. Any method known in the art may be used to manipulate the cell cycle phase. Methods to control the cell cycle phase include, but are not limited to, GO quiescence induced by contact inhibition of cultured cells, GO quiescence induced by removal of serum or other essential nutrient, GO quiescence induced by senescence, GO quiescence induced by addition of a specific growth factor; GO or GI quiescence induced by physical or chemical means such as heat shock, hyperbaric pressure or other treatment with a chemical, hormone, growth factor or other substance; S-phase control via treatment with a chemical agent which interferes with any. Point of the replication procedure; M-phase control via selection using fluorescence activated cell sorting, mitotic shake off, treatment with microtubule disrupting agents or any chemical which disrupts progression in mitosis (see also Freshney, R. I., “Culture of Animal Cells: A Manual of Basic Technique,” Alan R. Liss, Inc, New York (1983).

Methods for isolation of oocytes are well known in the art. Essentially, this can comprise isolating oocytes from the ovaries or reproductive tract of a pig. A readily available source of pig oocytes is slaughterhouse materials. For the combination of techniques such as genetic engineering, nuclear transfer and cloning, oocytes must generally be matured in vitro before these cells can be used as recipient cells for nuclear transfer, and before they can be fertilized by the sperm cell to develop into an embryo. This process generally requires collecting immature (prophase I) oocytes from mammalian ovaries, e.g., bovine ovaries obtained at a slaughterhouse, and maturing the oocytes in a maturation medium prior to fertilization or enucleation until the oocyte attains the metaphase II stage, which in the case of bovine oocytes generally occurs about 18-24 hours post-aspiration. This period of time is known as the “maturation period”.

A metaphase II stage oocyte can be the recipient oocyte, at this stage it is believed that the oocyte can be or is sufficiently “activated” to treat the introduced nucleus as it does a fertilizing sperm. Metaphase II stage oocytes, which have been matured in vivo have been successfully used in nuclear transfer techniques. Essentially, mature metaphase II oocytes can be collected surgically from either non-superovulated or superovulated porcine 35 to 48, or 39-41, hours past the onset of estrus or past the injection of human chorionic gonadotropin (hCG) or similar hormone.

After a fixed time maturation period, which ranges from about 10 to 40 hours, and preferably about 16-18 hours, the oocytes can be enucleated. Prior to enucleation the oocytes can be removed and placed in appropriate medium, such as HECM containing 1 milligram per milliliter of hyaluronidase prior to removal of cumulus cells. The stripped oocytes can then be screened for polar bodies, and the selected metaphase II oocytes, as determined by the presence of polar bodies, are then used for nuclear transfer. Enucleation follows.

Enucleation can be performed by known methods, such as described in U.S. Pat. No. 4,994,384. For example, metaphase II oocytes can be placed in either HECM, optionally containing 7.5 micrograms per milliliter cytochalasin B, for immediate enucleation, or can be placed in a suitable medium, for example an embryo culture medium such as CR1aa, plus 10% estrus cow serum, and then enucleated later, preferably not more than 24 hours later, and more preferably 16-18 hours later.

Enucleation can be accomplished microsurgically using a micropipette to remove the polar body and the adjacent cytoplasm. The oocytes can then be screened to identify those of which have been successfully enucleated. One way to screen the oocytes is to stain the oocytes with 1 microgram per milliliter 33342 Hoechst dye in HECM, and then view the oocytes under ultraviolet irradiation for less than 10 seconds. The oocytes that have been successfully enucleated can then be placed in a suitable culture medium, for example, CR1 aa plus 10% serum.

A single mammalian cell of the same species as the enucleated oocyte can then be transferred into the perivitelline space of the enucleated oocyte used to produce the NT unit. The mammalian cell and the enucleated oocyte can be used to produce NT units according to methods known in the art. For example, the cells can be fused by electrofusion. Electrofusion is accomplished by providing a pulse of electricity that is sufficient to cause a transient breakdown of the plasma membrane. This breakdown of the plasma membrane is very short because the membrane reforms rapidly. Thus, if two adjacent membranes are induced to breakdown and upon reformation the lipid bilayers intermingle, small channels can open between the two cells. Due to the thermodynamic instability of such a small opening, it enlarges until the two cells become one. See, for example, U.S. Pat. No. 4,997,384 by Prather et al. A variety of electrofusion media can be used including, for example, sucrose, mannitol, sorbitol and phosphate buffered solution. Fusion can also be accomplished using Sendai virus as a fusogenic agent (Graham, Wister Inot. Symp. Monogr., 9, 19, 1969). Also, the nucleus can be injected directly into the oocyte rather than using electroporation fusion. See, for example, Collas and Barnes, Mol. Reprod. Dev., 38:264-267 (1994). After fusion, the resultant fused NT units are then placed in a suitable medium until activation, for example, CR1aa medium. Typically activation can be effected shortly thereafter, for example less than 24 hours later, or about 4-9 hours later.

The NT unit can be activated by any method that accomplishes the desired result. Such methods include, for example, culturing the NT unit at sub-physiological temperature, in essence by applying a cold, or actually cool temperature shock to the NT unit. This can be most conveniently done by culturing the NT unit at room temperature, which is cold relative to the physiological temperature conditions to which embryos are normally exposed. Alternatively, activation can be achieved by application of known activation agents. For example, penetration of oocytes by sperm during fertilization has been shown to activate prefusion oocytes to yield greater numbers of viable pregnancies and multiple genetically identical pigs after nuclear transfer. Also, treatments such as electrical and chemical shock can be used to activate NT embryos after fusion. See, for example, U.S. Pat. No. 5,496,720, to Susko-Parrish, et al. Additionally, activation can be effected by simultaneously or sequentially by increasing levels of divalent cations in the oocyte, and reducing phosphorylation of cellular proteins in the oocyte. This can generally be effected by introducing divalent cations into the oocyte cytoplasm, e.g., magnesium, strontium, barium or calcium, e.g., in the form of an ionophore. Other methods of increasing divalent cation levels include the use of electric shock, treatment with ethanol and treatment with caged chelators. Phosphorylation can be reduced by known methods, for example, by the addition of kinase inhibitors, e.g., serine-threonine kinase inhibitors, such as 6-dimethyl-aminopurine, staurosporine, 2-aminopurine, and sphingosine. Alternatively, phosphorylation of cellular proteins can be inhibited by introduction of a phosphatase into the oocyte, e.g., phosphatase 2A and phosphatase 2B.

The activated NT units can then be cultured in a suitable in vitro culture medium until the generation of cell colonies. Culture media suitable for culturing and maturation of embryos are well known in the art. Examples of known media, which can be used for embryo culture and maintenance, include Ham's F-10+10% fetal calf serum (FCS), Tissue Culture Medium-199 (TCM-199)+10% fetal calf serum, Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's Phosphate Buffered Saline (PBS), Eagle's and Whitten's media.

Afterward, the cultured NT unit or units can be washed and then placed in a suitable media contained in well plates which preferably contain a suitable confluent feeder layer. Suitable feeder layers include, by way of example, fibroblasts and epithelial cells. The NT units are cultured on the feeder layer until the NT units reach a size suitable for transferring to a recipient female, or for obtaining cells which can be used to produce cell colonies. Preferably, these NT units can be cultured until at least about 2 to 400 cells, more preferably about 4 to 128 cells, and most preferably at least about 50 cells.

Activated NT units can then be transferred (embryo transfers) to the oviduct of an female pigs. In one embodiment, the female pigs can be an estrus-synchronized recipient gilt. Crossbred gilts (large white/Duroc/Landrace) (280-400 lbs) can be used. The gilts can be synchronized as recipient animals by oral administration of 18-20 mg ReguMate (Altrenogest, Hoechst, Warren, N.J.) mixed into the feed. Regu-Mate can be fed for 14 consecutive days. One thousand units of Human Chorionic Gonadotropin (hCG, Intervet America, Millsboro, Del.) can then be administered i.m. about 105 h after the last Regu-Mate treatment. Embryo transfers of the can then be performed about 22-26 h after the hCG injection. In one embodiment, the pregnancy can be brought to term and result in the birth of live offspring. In another embodiment, the pregnancy can be 5 terminated early and embryonic cells can be harvested.

The methods for embryo transfer and recipient animal management in the present invention are standard procedures used in the embryo transfer industry. Synchronous transfers are important for success of the present invention, i.e., the stage of the NT embryo is in synchrony with the estrus cycle of the recipient female. See, for example, Siedel, G. E., Jr. “Critical review of embryo transfer procedures with cattle” in Fertilization and Embryonic Development in Vitro (1981) L. Mastroianni, Jr. and J. D. Biggers, ed., Plenum Press, New York, N.Y., page 323.

VIII. Porcine Animals, Organs, Tissues, Cells and Cell Lines

The present invention provides viable porcine in which both alleles of the CMP-Neu5Ac hydroxylase gene have been inactivated. The invention also provides organs, tissues, and cells derived from such porcine, which are useful for xenotransplantation.

In one embodiment, the invention provides porcine organs, tissues and/or purified or substantially pure cells or cell lines obtained from pigs that lack any expression of functional CMP-Neu5Ac hydroxylase.

In one embodiment, the invention provides organs that are useful for xenotransplantation. Any porcine organ can be used, including, but not limited to: brain, heart, lungs, glands, brain, eye, stomach, spleen, pancreas, kidneys, liver, intestines, uterus, bladder, skin, hair, nails, ears, nose, mouth, lips, gums, teeth, tongue, salivary glands, tonsils, pharynx, esophagus, large intestine, small intestine, rectum, anus, pylorus, thyroid gland, thymus gland, suprarenal capsule, bones, cartilage, tendons, ligaments, skeletal muscles, smooth muscles, blood vessels, blood, spinal cord, trachea, ureters, urethra, hypothalamus, pituitary, adrenal glands, ovaries, oviducts, uterus, vagina, mammary glands, testes, seminal vesicles, penis, lymph, lymph nodes and lymph vessels.

In another embodiment, the invention provides tissues that are useful for xenotransplantation. Any porcine tissue can be used, including, but not limited to: epithelium, connective tissue, blood, bone, cartilage, muscle, nerve, adenoid, adipose, areolar, bone, brown adipose, cancellous, muscle, cartaginous, cavernous, chondroid, chromaffin, dartoic, elastic, epithelial, fatty, fibrohyaline, fibrous, Gaingee, gelatinous, granulation, gut-associated lymphoid, Haller's vascular, hard hemopoietic, indifferent, interstitial, investing, islet, lymphatic, lymphoid, mesenchymal, mesonephric, mucous connective, multilocular adipose, myeloid, nasion soft, nephrogenic, nodal, osseous, osteogenic, osteoid, periapical, reticular, retiform, rubber, skeletal muscle, smooth muscle, and subcutaneous tissue.

In a further embodiment, the invention provides cells and cell lines from porcine animals that lack expression of functional alpha1,3GT. In one embodiment, these cells or cell lines can be used for xenotransplantation. Cells from any porcine tissue or organ can be used, including, but not limited to: epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, □hosphate cells, cumulus cells, epidermal cells, endothelial cells, Islets of Langerhans cells, pancreatic insulin secreting cells, pancreatic alpha-2 cells, pancreatic beta cells, pancreatic alpha-1 cells, blood cells, blood precursor cells, bone cells, bone precursor cells, neuronal stem cells, primordial stem cells, hepatocytes, keratinocytes, umbilical vein endothelial cells, aortic endothelial cells, microvascular endothelial cells, fibroblasts, liver stellate cells, aortic smooth muscle cells, cardiac myocytes, neurons, Kupffer cells, smooth muscle cells, Schwann cells, and epithelial cells, erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes, pancreatic islet cells, thyroid cells, parathyroid cells, parotid cells, tumor cells, glial cells, astrocytes, red blood cells, white blood cells, macrophages, epithelial cells, somatic cells, pituitary cells, adrenal cells, hair cells, bladder cells, kidney cells, retinal cells, rod cells, cone cells, heart cells, pacemaker cells, spleen cells, antigen presenting cells, memory cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, uterine cells, prostate cells, vaginal epithelial cells, sperm cells, testicular cells, germ cells, egg cells, leydig cells, peritubular cells, sertoli cells, lutein cells, cervical cells, endometrial cells, mammary cells, follicle cells, mucous cells, ciliated cells, nonkeratinized epithelial cells, keratinized epithelial cells, lung cells, goblet cells, columnar epithelial cells, dopaminergic cells, squamous epithelial cells, osteocytes, osteoblasts, osteoclasts, embryonic stem cells, fibroblasts and fetal fibroblasts. In a specific embodiment, pancreatic cells, including, but not limited to, Islets of Langerhans cells, insulin secreting cells, 48 alpha-2 cells, beta cells, alpha-1 cells from pigs that lack expression of functional alpha-1,3-GT are provided.

Nonviable derivatives include tissues stripped of viable cells by enzymatic or chemical treatment these tissue derivatives can be further processed via crosslinking or other chemical treatments prior to use in transplantation. In a preferred embodiment, the derivatives include extracellular matrix derived from a variety of tissues, including skin, urinary, bladder or organ submucosal tissues. Also, tendons, joints and bones stripped of viable tissue to include heart valves and other nonviable tissues as medical devices are provided.

Therapeutic Uses

The cells can be administered into a host in order in a wide variety of ways. Preferred modes of administration are parenteral, intraperitoneal, intravenous, intradermal, epidural, intraspinal, intrasternal, intra-articular, intra-synovial, intrathecal, intra-arterial, intracardiac, intramuscular, intranasal, subcutaneous, intraorbital, intracapsular, topical, transdermal patch, via rectal, vaginal or urethral administration including via suppository, percutaneous, nasal spray, surgical implant, internal surgical paint, infusion pump, or via catheter. In one embodiment, the agent and carrier are administered in a slow release formulation such as a direct tissue injection or bolus, implant, microparticle, microsphere, nanoparticle or nanosphere.

Disorders that can be treated by infusion of the disclosed cells include, but are not limited to, diseases resulting from a failure of a dysfunction of normal blood cell production and maturation (i.e., aplastic anemia and hypoproliferative stem cell disorders); neoplastic, malignant diseases in the hematopoietic organs (e.g., leukemia and lymphomas); broad spectrum malignant solid tumors of non-hematopoietic origin; autoimmune conditions; and genetic disorders. Such disorders include, but are not limited to diseases resulting from a failure or dysfunction of normal blood cell production and maturation hyperproliferative stem cell disorders, including aplastic anemia, pancytopenia, agranulocytosis, thrombocytopenia, red cell aplasia, Blackfan Diamond syndrome, due to drugs, radiation, or infection, idiopathic; hematopoietic malignancies including acute lymphoblastic (lymphocytic) leukemia, chronic lymphocytic leukemia, acute myclogenous leukemia, chronic myelogenous, leukemia, acute malignant myelosclerosis, multiple myeloma, polycythemia vera, agnogenic myelometaplasia, Waldenstrom's macroglobulinemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma; immunosuppression in patients with malignant, solid tumors including malignant melanoma, carcinoma of the stomach, ovarian carcinoma, breast carcinoma, small cell lung carcinoma, retinoblastoma, testicular carcinoma, glioblastoma, rhabdomyosarcoma, neuroblastoma, Ewing's sarcoma, lymphoma; autoimmune diseases including rheumatoid arthritis, diabetes type 1, chronic hepatitis, multiple sclerosis, systemic lupus erythematosus; genetic (congenital) disorders including anemias, familial aplastic, Fanconi's syndrome, dihydrofolate reductase deficiencies, formamino transferase deficiency, Lesch-Nyhan syndrome, congenital dyserythropoietic syndrome IIV, Chwachmann-Diamond syndrome, dihydrofolate reductase deficiencies, forinamino transferase deficiency, Lesch-Nyhan syndrome, congenital spherocytosis, congenital elliptocytosis, congenital stomatocytosis, congenital Rh null disease, paroxysmal nocturnal hemoglobinuria, G6PD (glucose □hosphate dehydrogenase) variants 1, 2, 3, pyruvate kinase deficiency, congenital erythropoietin sensitivity, deficiency, sickle cell disease and trait, thalassemia alpha, beta, gamma, met-hemoglobinemia, congenital disorders of immunity, severe combined immunodeficiency disease (SCID), bare lymphocyte syndrome, ionophore-responsive combined immunodeficiency, combined immunodeficiency with a capping abnormality, nucleoside phosphorylase deficiency, granulocyte actin deficiency, infantile agranulocytosis, Gaucher's disease, adenosine deaminase deficiency, Kostmann's syndrome, reticular dysgenesis, congenital Leukocyte dysfunction syndromes; and others such as osteoporosis, myelosclerosis, acquired hemolytic anemias, acquired immunodeficiencies, infectious disorders causing primary or secondary immunodeficiencies, bacterial infections (e.g., Brucellosis, Listerosis, tuberculosis, leprosy), parasitic infections (e.g., malaria, Leishmaniasis), fungal infections, disorders involving disproportionsin lymphoid cell sets and impaired immune functions due to aging, phagocyte disorders, Kostmann's agranulocytosis, chronic granulomatous disease, Chediak-Higachi syndrome, neutrophil actin deficiency, neutrophil membrane GP-180 deficiency, metabolic storage diseases, mucopolysaccharidoses, mucolipidoses, miscellaneous disorders involving immune mechanisms, Wiskott-Aldrich Syndrome, alpha lantirypsin deficiency, etc.

Diseases or pathologies include neurodegenerative diseases, hepatodegenerative diseases, nephrodegenerative disease, spinal cord injury, head trauma or surgery, viral infections that result in tissue, organ, or gland degeneration, and the like. Such neurodegenerative diseases include but are 10 not limited to, AIDS dementia complex; demyeliriating diseases, such as multiple sclerosis and acute transferase myelitis; extrapyramidal and cerebellar disorders, such as lesions of the ecorticospinal system; disorders of the basal ganglia or cerebellar disorders; hyperkinetic movement disorders, such as Huntington's Chorea and senile chorea; drug-induced movement disorders, such as those induced by drugs that block CNS dopamine receptors; hypokinetic movement disorders, such as Parkinson's disease; progressive supra-nucleo palsy; structural lesions of the cerebellum; spinocerebellar degenerations, such as spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiple systems degenerations (Mencel, Dejerine Thomas, Shi-Drager, and Machado-Joseph), systermioc disorders, such as Rufsum's disease, abetalipoprotemia, ataxia, telangiectasia; and mitochondrial multisystem disorder; demyelinating core disorders, such as multiple sclerosis, acute transverse myelitis; and disorders of the motor unit, such as neurogenic muscular atrophies (anterior horn cell degeneration, such as amyotrophic lateral sclerosis, infantile spinal muscular atrophy and juvenile spinal muscular atrophy); Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy body disease; Senile Dementia of Lewy body type; Parkinson's Disease, Wernicke-Korsakoff syndrome; chronic alcoholism; Creutzfeldt-Jakob disease; Subacute sclerosing panencephalitis hallefforden-Spatz disease; and Dementia pugilistica. See, e.g., Berkow et. al., (eds.) (1987), The Merck Manual, (15′) ed.), Merck and Co., Rahway, N.J.

Industrial Farming Uses

The present invention provides viable porcine for purposes of farming applications in which one or both alleles of the CMP-Neu5Ac hydroxylase gene have been inactivated. Inactivation of one or both alleles of the CMP-Neu5Ac hydroxylase gene can reduce the susceptibility of porcine animals to zoonotic diseases and infections in pigs such as, for example, E. coli, pig rotavirus, and pig transmissible gastroenteritis coronavirus, and any other zoonotic or enterotoxigenic organism that utilizes Neu5Gc in a host animal. The reduction in disease susceptibility allows greater economic realization of farming operations due to the ability to harvest more healthy animals, and the reduction of animal death due to enterotoxigenic organisms.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES Isolation of Nucleic Acids

Combination strategy of PCR-based methods was employed to identify the porcine CMP-Neu5Ac hydroxylase gene. Such PCR methods are well known in the art and described, for example, in PCR Technology, H. A. Erlich, ed., Stockton Press, London, 1989; PCR Protocols: A Guide to Methods and Applications, M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, eds., Academic Press, Inc., New York, 1990.

Total RNA was extracted from an adult porcine (Great Yorkshire) spleen using Trizol reagent (Gibco, Grand Island, N.Y.). After treatment with Dnase I (Ambion, Inc., Austin, Tex.), poly A⁺ RNA was separated using the Dynabeads mRNA Purification Kit (Dynal, Oslo, Norway). To identify the 5′- or 3′-end of porcine CMP-Neu5Ac Hydroxylase gene, 5′- or 3′-RACE (rapid amplification of cDNA ends) procedures were performed using Marathon™ cDNA Amplification kit (Clontech). To identify exon-intron boundaries, or 5′- or 3′-flanking region of the transcripts, porcine GenomeWalker™ libraries were constructed using Universal GenomeWalker™ Library kit (Clontech). Gene-specific and nested primer pairs were designed from the partial cDNA sequence provided by GenBank Accession #A59058.

Determination of cDNA and Genomic CMP-Neu5Ac Hydroxylase Sequence

5′- or 3′-RACE analysis: To identify the 5′ and 3′ ends of porcine CMP-Neu5Ac hydroxylase gene transcripts, 5′- and 3′-RACE procedures were performed using the Marathon cDNA Amplification Kit (Clontech) with poly A+ RNA isolated from adult porcine spleen as a template. First strand cDNA synthesis from 1 ug of poly A+ RNA was accomplished using 20 U of AMV-RT and 1 pmol of the supplied cDNA Synthesis Primer by incubating at 48° C. for 2 hours. Second strand cDNA synthesis involved incubating the entire first strand reaction with a supplied enzyme cocktail composed of Rnase H, E. coli DNA polymerase I, and E. coli DNA polymerase I, and E. coli DNA ligase at 16° C. for 1.5 hr. After blunting of the double stranded cDNA ends by T4 DNA polymerase, the supplied Marathon cDNA Adapters were ligated to an aliquot of purified, double-stranded cDNA. Dilution of the adapter-ligated product in 10 mM ticme-KOH/0.1 mM EDTA buffer provided with the kit readied the cDNA for PCR amplification.

To obtain the 5′- and 3′-most sequences of the porcine CMP-Neu5Ac hydroxylase gene transcripts, provided Marathon cDNA Amplification primer sets were paired with gene-specific and nested gene-specific primers based on the sequence provided by GenBank accession number A59058. These primer sets are provided for in Table 13. By this method, oligonucleotide primers based on the sequence contained in Genbank accession number A59058 are oriented in the 3′ and 5′ directions and are used to generate overlapping PCR fragments. These overlapping 3′ and 5′ products are combined to produce an intact full-length cDNA. This method is described, for example, in Innis, et al., supra; and Frohman et al., Proc. Natl. Acad. Sci., 85:8998, 1988, and further described, for example, in U.S. Pat. No. 4,683,195.

Genome Walking analysis: To identify exon-intron boundaries, or 5′- or 3′-flanking region of the porcine CMP-Neu5Ac hydroxylase transcripts, porcine GenomeWalker™ libraries were constructed using the Universal GenomeWalker™ Library Kit (Clontech, Palo Alto, Calif.).

Briefly, five aliquots of porcine genomic DNA were separately digested with a single blunt-cutting restriction endonuclease (DraI, EcoRV, PvuII, ScaI, or StuI). After phenol-chloroform extraction, ethanol precipitation, and resuspension of the restricted fragments, a portion of each digested aliquot was used in separate ligation reaction with the GenomeWalker adapters provided with the kit. This process created five libraries for use in the PCR based cloning strategy. Primer pairs identified in Table 13 were used in a genome walking strategy. Either eLON-Gase or TaKaRa LA Taq (Takara Shuzo Co., Ltd., Shiga, Japan) enzyme was used for PCR in all GenomeWalker experiments as well as for direct long PCR of genomic DNA. The thermal cycling conditions recommended by the manufacturer were employed in all GenomeWalker-PCR experiments on a Perkin Elmer Gene Amp System 9600 or 9700 thermocycler.

TABLE 13 Primers Used in PCR Strategies Primer PCR Set Strategy Sequence XA 3′-RACE/ 5′-CATGGACCTCAAGCTGGGGGACAAGA-3′ Genome Walking XB 3′-RACE/ 5′-GTGTTCGACCCTTGGTTAATCGGTCCTG-3′ Genome Walking XM 5′-RACE/ 5′-CAGGACCGATTAACCAAGGGTCGAACAC-3′ Genome Walking XN 5′-RACE/ 5′-TCTTGTCCCCCAGCTTGAGGTCCATG-3′ Genome Walking

Subcloning and sequencing of amplified products: PCR products amplified from genomic DNA, GeneWalker-PCR (Clontech), and 5′-3′-RACE wre gel-purified using the Qiagen Gel Extraction Kit (Qiagen, Valencia, Calif.), if necessary, then subcloned into the pCR11 vector provided with the Original TA Cloning Kit (Invitrogen, Carlsbad, Calif.). Plasmid DNA minipreps of pCR11-ligated inserts were prepared with the QIAprep Spin Miniprep Kit (Qiagen) as directed. Automated fluorescent sequencing of cloned inserts was performed using an ABI 377 Automated Sequence Analyzer (Applied Biosystems, Inc., Foster City, Calif.) with either the dRhodamine or BigDye Terminator Cycle Sequencing Kits (Applied Biosystems) primed with T7 and SP6 promoter primers or primers designed from internal insert sequences.

Primer Synthesis: All oligonucleotides used as primers in the various PCR-based methods were synthesized on an ABI 394 DNA Synthesizer (Applied Biosystems, Inc., Foster City Calif.) using solid phase synthesis and phosphoramidite nucleoside chemistry, unless otherwise stated.

Analysis of Transcription Factor Binding Sites

Analysis of possible transcription factors binding sites were performed using 228 bp of exon 1 sequence and 601 bp upstream of exon 1. The sequences were screened using “MatInspector” software available in www.genomatix.de. The sequences contain binding sites for the following transcription factors: MZF1, ETSF, SF1, CMYB, MEF2, NMP4, BRN2, AP1, GAT1, SATB1, ATF, USF, WHN, ZF5, NFκB, MOK2, NFY, MYCMAX, ZF5. See FIG. 4.

Construction of Porcine CMP-Neu5Ac Hydroxylase Homologous Recombination Targeting Vectors

CMP-Neu5Ac hydroxylase knock-out target vector: A vector targeting Exon 6 of the porcine CMP-Neu5Ac Hydroxylase gene for knockout can be constructed. In a first step, a portion of Intron 6 is amplified by PCR for use as a 3′-arm of the targeting vector utilizing primers such as pDH3 (5′-CTCCTGGAAGCTTCTGTCAAGACGAAC-3′) and pDH4 (5′-GCCTGATACACAGTGCTGTGCAATGGT-3′) (see FIG. 5). The amplified PCR product of approximately 3.7 kb can be inserted into the pCRII vector after restriction enzyme digestion utilizing EcoRI and ApaI. See FIG. 6.

Following the insertion of the 3′-arm, a portion of Intron 5 can be amplified by PCR for use as a 5′-arm in the targeting vector utilizing primers such as pDH1 (5′-ACCACCCAAGTCTGGAATCTTCTTACACT-3′) and pDH2 (5′-GACTCTCATACAAAAGCTAAGCTGGGTAAG-3′) (see FIG. 5). Following this initial amplification, successive PCR amplifications can be performed to introduce an EcoNI restriction site into the 3′ portion of the 5′-arm utilizing primers such as pDH1 in conjunction with primers such as pDH2a (5′-GACTCTCATACAAAACCTAAGCTGGGTAAG-3′), pDH2b (5′-GACTCTCATACAAAACCTAGGCTGGGTAAG-3′), and pDH2c (5′-GACTCTCATACAAAACCTAGGCTAGGTAAG-3′), respectively (see FIG. 5). The amplified PCR product of approximately 2.6 kb containing the engineered EcoNI site can be restriction enzyme digested using ApaI and EcoNI, and inserted into the pCRII vector containing the previously inserted 3′-arm (See FIG. 7), generating a targeting vector (pDHΔex6) containing an approximate 6.3 kb porcine CMP-Neu5Ac hydroxylase targeting sequence (see FIG. 8).

EGFP knock-in target vector: pDHΔex6 can be further modified by an in-frame insertion of an enhanced green fluorescent protein sequence at the terminal 3′ end of Exon 6 of the porcine CMP-Neu5Ac hydroxylase gene. In a first step, a portion of Intron 5 and a portion of Exon 6 of the porcine CMP-Neu5Ac hydroxylase gene can be amplified by PCR utilizing primers such as pDH5 (5′-CCTTATACTGGCCCCAATTGGATCTTAC-3′) and pDH6 (5′-CCTTATACTGGCCCCAATTGGATCTTAC-3′) (see FIG. 9), and inserted into a vector (pIRES-EGFP) containing the EGFP and a poly A tail following restriction enzyme digestion with MunI and EcoRv. Following insertion, PCR amplification can be performed on the pIRES-EGFP vector containing the insertion utilizing primer such as pDH7 (5′-CTTACCTAGCCTAGGTTTTGTATGAGAGTC-3′) and pDH8 (5′-GACAAACCACAATTGGAATGCACTCGAG-3′) (see FIG. 9). The PCR amplified product can be restriction enzyme digested using EcoNI and MunI and inserted into the previously constructed pDHΔex6 targeting vector (see FIG. 10). The resultant targeting vector (pDHΔex6-EGFP) is illustrated in FIG. 11.

Production of Porcine CMP-Neu5Ac Hydroxylase Deficient Fetal Fibroblast Cells

Fetal fibroblast cells are isolated from 10 fetuses of the same pregnancy at day 33 of gestation. After removing the head and viscera, fetuses are washed with Hanks' balanced salt solution (HBSS; Gibco-BRL, 1 5 Rockville, Md.), placed in 20 ml of HBSS, and diced with small surgical scissors. The tissue is pelleted and resuspended in 50-ml tubes with 40 ml of DMEM and 100 U/ml collagenase (Gibco-BRL) per fetus. Tubes are incubated for 40 min in a shaking water bath at 37 C. The digested tissue is allowed to settle for 3-4 min and the cell-rich supernatant is transferred to a new 50-ml tube and pelleted. The cells are then resuspended in 40 ml of DMEM containing 10% fetal calf serum (FCS), 1X nonessential amino acids, 1 mM sodium pyruvate and 2 ng/ml bFGF, and seeded into 10 cm. dishes. For transfections, 10 μg of linearized pDHΔex6EGFP vector is introduced into 2 million cells using lipofectamine 2000 (Carlsbad, Calif.) following manufacturer's guidelines. Forty-eight hours after transfection, the transfected cells are seeded into 48-well plates at a density of 2,000 cells per well and grown to confluence. Following confluence, cells are sorted via Fluorescent Activated Cell Sorting (FACS) (FACSCalibur, Becton Dickenson, San Jose, Calif.), wherein only cells having undergone homologous recombination and expressing the EGFP are selected (see, for example, FIG. 13).

Selected cells are then reseeded, and grown to confluency. Once confluency is reached, several small aliquots are frozen back for future use, and the remainder are utilized for PCR and Southern Blot verification of homologous recombination. The putative targeted clones can be screened by PCR across the Exon 6/EGFP insert utilizing a primer complimentary to the EGFP sequence and a primer complimentary to a sequence outside the vector as the antisense primer. The PCR products can be analyzed by Southern Blotting using an EGFP probe to identify the positive clones by the presence of the expected band from the targeted allele.

Generation of Cloned Pigs Using Heterologous CMP-Neu5Ac Hydroxylase Deficient Fetal Fibroblasts as Nuclear Donors

Preparation of cells for Nuclear Transfer: Donor cells are genetically manipulated to produce cells heterozygous for porcine CMP-Neu5Ac hydroxylase as described generally above. Nuclear transfer can be performed by methods that are well known in the art (see, e.g., Dai et al., Nature Biotechnology 20: 251255, 2002; and Polejaeva et al., Nature 407:86-90, 2000), using EGFP selected porcine fibroblasts as nuclear donors that are produced as described in detail hereinabove.

Oocytes can be isolated from synchronized super ovulated sexually mature Large-White X Landacre outcross gilts as described, for example, in 1. Polejaeva et al. Nature 407: 505 (2000). Donor cells are synchronized in presumptive G0/G1 by serum starvation (0.5%) between 24 to 120 hours. Oocytes enucleation, nuclear transfer, electrofusion, and electroactivation can be performed as essentially described in, for example, A. C. Boquest et al., Biol. Reproduction 68: 1283 (2002). Reconstructed embryos can be cultured overnight and can be transferred to the oviducts of asynchronous (−1 day) recipients. Pregnancies can be confirmed and monitored by real-time ultrasound.

Breeding of heterozygous CMP-Neu5Ac hydroxylase single knockout (SKO) male and female pigs can be performed to establish a miniherd of double knockout (DKO) pigs.

Verification of CMP-Neu5Ac Hydroxylase Deficient Pigs

Following breeding of the single knockout male and female pigs, verification of double knockout pigs is performed. Fibroblasts from the offspring are incubated with 1 μg of anti-N-glycolyl GM2 monoclonal antibody MK2-34 (Seikagaku Kogyo, JP) on ice for 30 minutes. FITC conjugated goat-anti-mouse IgG is added to the cells and antibody binding indicating the presence or absence of Neu5GC, and thus, an indication of the presence or absence of active CMP-Neu5Ac hydroxylase, is detected by flow cytometry (FACSCalibur, Becton Dickenson, San Jose, Calif.). 

1-66. (canceled)
 67. A targeting vector for homologous recombination in a somatic cell comprising a marker sequence and a sequence homologous to a porcine genomic CMP-N-acetylneuraminic-acid (CMP-Neu5Ac) hydroxylase sequence, wherein said homologous sequence is sufficient to provide targeted insertion of the selectable marker sequence into a target CMP-Neu5Ac gene in a host.
 68. The targeting vector of claim 67 wherein said vector comprises: i. a first nucleotide sequence comprising at least 17 contiguous nucleotides of the porcine genomic CMP-Neu5Ac hydroxylase sequence; ii. a second nucleotide sequence comprising at least 17 contiguous nucleotides of the porcine genomic CMP-Neu5Ac hydroxylase sequence; and iii. a marker sequence; wherein said first and second nucleotide sequences do not overlap.
 69. The targeting vector of claim 68 wherein the selectable marker gene is green fluorescent protein.
 70. The targeting vector of claim 68 wherein the first nucleotide sequence represents a 5′ recombination arm.
 71. The targeting vector of claim 68 wherein the second nucleotide sequence represents a 3′ recombination arm.
 72. The targeting vector of claim 68 wherein the first or second nucleotide sequence is homologous to Intron 5 of porcine genomic CMP-Neu5Ac hydroxylase sequence.
 73. The targeting vector of claim 68 wherein the first or second nucleotide sequence is homologous to Intron 6 of the porcine genomic CMP-Neu5Ac hydroxylase sequence.
 74. The targeting vector of claim 68 wherein the first or second nucleotide sequence comprises at least 50 contiguous nucleotides of the porcine genomic CMP-Neu5Ac hydroxylase sequence.
 75. The targeting vector of claim 68 wherein the first or second nucleotide sequence comprises at least 100 contiguous nucleotides of the porcine genomic CMP-Neu5Ac hydroxylase sequence.
 76. The targeting vector of claim 68 wherein the first or second nucleotide sequence comprises at least 150 contiguous nucleotides of the porcine genomic CMP-Neu5Ac hydroxylase sequence.
 77. A cell transfected with the targeting vector of claim
 67. 78. The cell of claim 77 wherein at least one allele of a porcine CMP-Neu5Ac gene has been rendered inactive via homologous recombination.
 79. A porcine animal comprising the cell of claim
 78. 80. The animal of claim 79 wherein at least one allele of a porcine CMP-Neu5Ac hydroxylase gene has been rendered inactive via homologous recombination.
 81. An organ obtained from the animal of claim
 80. 82. A tissue obtained from the animal of claim
 80. 83. The organ of claim 81 wherein the organ is selected from the group consisting of heart, lung, kidney and liver.
 84. A method to produce genetically modified cells comprising: (a) transfecting a porcine cell with the targeting vector of claim 67; and (b) selecting a transfected cell in which at least one allele of a porcine CMP-N-acetylneuraminic-acid (CMP-Neu5Ac) hydroxylase gene has been rendered inactive.
 85. The method of claim 84 further comprising: (c) transferring the nucleus of the selected transfected cell into an enucleated oocyte to produce an embryo; and (d) allowing the embryo to develop into a non-human animal wherein at least one allele of a porcine CMP-Neu5Ac hydroxylase gene has been rendered inactive. 